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Full text of "AOAC: Official Methods of Analysis, 1980"

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By Authority Of 

THE UNITED STATES OF AMERICA 

Legally Binding Document 



By the Authority Vested By Part 5 of the United States Code § 552(a) and 
Part 1 of the Code of Regulations § 51 the attached document has been duly 
INCORPORATED BY REFERENCE and shall be considered legally 
binding upon all citizens and residents of the United States of America. 
HEED THIS NOTICE : Criminal penalties may apply for noncompliance. 




Document Name: AOAC: Official Methods of Analysis, 1980 



CFR Section(s) : 2 1 CFR 131.1 50(c) 



Standards Body: AOAC International 



OFFICIAL 

METHODS OF ANALYSIS 

OF THE 

ASSOCIATION OF OFFICIAL 
ANALYTICAL CHEMISTS 



William Horwitz, Editor 



THIRTEENTH EDITION, 1980 



Published by the 

Association of Official Analytical Chemists 

PO Box 540, Benjamin Franklin Station 

Washington, DC 20044 



Direct inquiries related to the scientific content of Official Methods of Analysis to: 

Editor, Official Methods of Analysis 
Association of Official Analytical Chemists 
Box 540, Benjamin Franklin Station 
Washington, DC 20044 USA 



Direct inquiries related to the procurement of Official Methods of Analysis, supplements (Changes in Methods), Journal of the 
AOAC, or other AOAC publications to: 

Assistant Business Manager, Publications 

Association of Official Analytical Chemists 

1111 N 19th Street (Suite 210) 

Arlington, VA 22209 USA (Telephone: 703-522-3032) 



Copyright 1920, 1925, 1931, 1936, 1940, 1945, 1950, 1955, 1960, 1965, by the 

Association of Official Agricultural Chemists 

and 1970, 1975, 1980 by the 

Association of Official Analytical Chemists 



The methods of the Association were also copyrighted in 1916, when they 

were published in the Journal of the Association of 

Official Agricultural Chemists 

Library of Congress Catalog Card Number: 20-21343 
ISBN 0-935584-14-5 

A copy of the 13th edition of this publication is on file with the Office of the Federal Register. U. S. Government Agencies may apply 
to the Director of the Office of the Federal Register for approval to incorporate this edition by reference in their regulations. The 
procedures that Federal agencies must follow in applying for the Director's approval are in Title 1, Part 51 of the Code of Federal 
Regulations. 

Composed by 

Monotype Composition Company, Inc. 

Baltimore, Maryland 

Printed and bound by 

George Banta Company, Inc. 

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ii 



Preface to Thirteenth Edition 



The most noticeable physical change in this thirteenth edition 
of Official Methods of Analysis of the Association of Official 
Analytical Chemists ('The Book of Methods'') is its size. A survey 
of users of the Book of Methods revealed an overwhelming 
desire to maintain the compendium as a single volume, The 
easiest way to do this was to increase the size of the page. 
Users also expressed a desire for a system that will keep the 
same reference number for a given method from edition to 
edition. A practical system that will maintain the continuity of 
the numbering system and the organizational structure of the 
methods within a chapter has not yet been devised. 

Approximately 175 new methods have been added during the 
current five year period (1974-1978); 83 methods have been 
deleted, replaced, or surplused. The approval of an average of 
only 35 new methods per year represents a marked decline from 
the 70 per year of the four year period of the previous edition 
(1970-1973) and the 50 per year of the five year period of the 
tenth edition (1965-1969). The decline is undoubtedly the result 
of a number of factors. Chief among them are the greater 
complexity of modern methods, requiring a large investment in 
resources that is not readily mobilized to fit an Associate 
Referee's schedule; and the fact that government agencies are 
attempting to obtain compliance, especially of the newer stat- 
utes, by promulgation of regulations and by auditing rather than 
by laboratory examinations. 

The greatest activity, as measured by approval of new meth- 
ods, is in the field of pesticide formulations, partly as a result 
of the active implementation of the cooperative agreement 
with the Collaborative International Pesticide Analytical Council 
(CIPAC). Other active areas include extraneous materials, vita- 
mins and other nutrients, dairy products, and microbiological 
methods. In fact, examination for and by biological constituents 
(filth, microbiological assays, and examination for food-borne 
pathogens) comprises approximately 17% of the new methods 
adopted. 

A comparison of the types of methods adopted between this 
and the previous edition reveals that gas-liquid chromatography 
has overtaken spectrophotometry in its various forms (visible, 
ultraviolet, and fluorescent) as the most predominant quanti- 
tative technique in Official Methods, High pressure (or perform- 
ance) liquid chromatography has spurted from none to 6% of 
the adopted methods within the relatively short period of five 
years. Electrometric methods (potentiometric, polarographic, 
and ion-selective) are now sufficiently numerous to deserve a 
separate category. Infrared methods are no longer a major 
factor in quantitation; they are now mainly used for identification 
and confirmation. A comparison of the present and previous 
editions is presented in Table 1. The figures given are only 
rough approximations because of the arbitrariness often re- 
quired in classifying a method and in deciding when a new 
method or revision is sufficiently independent to warrant con- 
tributing to the statistics. 



Table 1. Classification of new methods approved by the AOAC 
in the thirteenth and twelfth editions 





13th edition 


12th edition 


Method classification 


(1980) 


(1975) 


Gas-liquid chromatography 


18% 


14% 


Spectrophotometry 


16 


20 


Titrimetric 


10 


7 


Filth, isolation 


8 


14 


Automated 


7 


5 


Biological and microbiological 


9 


8 


High pressure liquid chromatography 


6 


— 


Atomic absorption 


4 


9 


Electrometric 


5 


— 


Chromatography (thin layer, etc.) 


3 


3 


Gravimetric 


2 


6 


Miscellaneous (physical, qualitative, 






etc.) 


11 


13 



The most interesting new collaboratively studied method is 
the mass spectrometric method for the detection of adulteration 
of honey with high fructose corn sirup. Corn sirups (from a 
monocotyledonous plant) have a distinctly different 13 C/ 12 C ratio 
than sirups from most dicotyledonous plants, which are the 
source of most honeys. The isotope ratio mass spectrometer 
required forthis determination is a highly specialized instrument, 
even in the field of mass spectrometry. Despite the rarity of the 
instrumentation, sufficient laboratories participated in the col- 
laborative study to establish the reproducibility of the method. 

A problem that has arisen is how to handle the numerous 
individual instruments of diverse design and manufacture that 
have been developed to automate a particular determination. 
Even if this problem is solved by incorporating all the available 
instruments into the initial collaborative study, the problem 
returns with the first "new and improved" modification. Different 
instrument designs and their subsequent modifications have 
been handled in the infrared determination of milk constituents 
by providing performance specifications which must be met by 
the basic instrument in general, when compared to a reference 
method or reference sample. In addition, the user must satisfy 
himself that his particular instrument also meets the perform- 
ance specifications by frequent comparisons with the reference 
method or sample. This requirement eliminates the need for 
repeated collaborative studies every time a manufacturer rede- 
signs or modifies his basic equipment, and in addition provides 
a continuous quality control technique on the performance of 
the instrument, method, and laboratory. 

It should not be overlooked that automation is not confined 
to physical and chemical determinations. The microbiological 
chapter contains three collaboratively studied instrumental 
methods for somatic cell count and an instrumental method for 
distributing a liquid sample for plate counting. Biochemical 
diagnostic kits have also been evaluated collaboratively for their 



effectiveness in identifying Salmonella and other enteric isolates. 
A criticism that is often leveled at the AOAC validation 
mechanism is that it is too slow to keep up with the pace of 
requirements for methods by regulatory agencies and the 
regulated industry. This objection overlooks the point that the 
speed with which a method is validated is almost completely in 
the hands of those needing the validated method, rather than 
in the hands of the AOAC. The method must be tested for 
ruggedness, and the directions tested for clarity; samples must 
be prepared and distributed; laboratories must analyze the 
samples and report the results. Dubious results must be inves- 
tigated and occasionally samples must be reanalyzed. The data 
must be tabulated and analyzed, and a report written. There is 
no way to short-cut the process of obtaining intra- and interlab- 
oratory variability except to conduct the necessary experimental 
work and perform the necessary statistical calculations. Only 
then is the recommendation subject to the time restrictions of 
the AOAC mechanism. To ensure that the AOAC mechanism of 
annual approval is not holding up use of needed methods, a 
new temporary class of methods has been introduced entitled 
"Interim First Action." These are methods which, together with 
their supporting studies, have been received between annual 
meetings and have been sent through the customary reviewing 
procedure. To be designated Interim First Action, a method 
must have been approved by the appropriate Referee and 
Subcommittee, and by the Chairman of the Committee on 
Official Methods. The method only lacks the formal vote by 
those AOAC members having regulatory authority over the 
commodity involved. Such formal approval is usually provided 
at the next annual meeting, at which time the method becomes 



"First Action." Approximately five interim methods are approved 
annually. Several such methods appear in this volume, depend- 
ing upon the time of their submission and review in relation to 
the editorial status of the chapter to which they are assigned. 
The current status of these Interim First Action methods, as well 
as the status of any method, can be found by consulting the 
latest cumulative index to Changes in Methods, which appears 
as the final pages of each March issue of the Journal. 

For those users of AOAC methods who may not be familiar 
with the procedures by which a method is included in this 
volume, we are reprinting a paper prepared for the Joint 
International Symposium, "The Harmonization of Collaborative 
Studies," held in London, England 9-10 March 1978. The 
document was drafted by the AOAC Committee on Collaborative 
Studies: Elwyn D. Schall, Chairman; Charles W. Gehrke, William 
Horwitz, Anthony J. Malonski, James P. Minyard, Jr., Forrest W. 
Quackenbush, and Ernest S. Windham. This paper appeared in 
Analytical Chemistry (March 1978), 50, 337A-340A. 

The publication of this "Book of Methods" is possible only 
because of the extensive cooperation of thousands of analysts 
who have volunteered to direct, participate in, and review the 
numerous collaborative studies that form the basis for inclusion 
of the several thousand methods that appear herein. Special 
recognition is due to Mrs. Betty Johnson who has prepared an 
entirely new index for this edition. 



28 September, 1979 



William Horwitz, Ph.D. 
Editor 



Abstract from 

Preface to First Edition 



"In presenting this revision of the official and tentative methods of analysis of the Association of Official Agricultural Chemists, it 
is appropriate to give a brief statement of the organization of the Association, its purpose, and the procedure by which the methods 
are adopted. 

"Membership in the Association is institutional and includes the State Departments of Agriculture, the State Agricultural Colleges 
and Experiment Stations, the Federal Department of Agriculture, and the Federal, State, and City offices charged with the enforcement 
of food, feed, drug, fertilizer, insecticide and fungicide control laws. 

"The Association was founded at Philadelphia, Pa., September 9, 1884, by the following representative agricultural chemists of that 
time, the organization being the result of a series of informal meetings held the immediately preceding years: 

"Prof. H. W. Wiley, Chemist of the Department of Agriculture, Washington, D.C. 

Mr. Clifford Richardson, Assistant Chemist of the Department of Agriculture, Washington, D.C. 

Mr. Philip E. Chazal, State Chemist of South Carolina. 

Dr. Chas. W. Dabney, Jr., State Chemist of North Carolina. 

Dr. W. J. Gascoyne, State Chemist of Virginia. 

Dr. E. H. Jenkins, Connecticut Experiment Station. 

Prof. John A. Meyers, State Chemist of Mississippi. 

Prof. H. C. White, State Chemist of Georgia. 

Mr. C. DeGhequier, Secretary National Fertilizer Association. 

Dr. Schumann, Dr. Lehmann, Mr. Gaines and others." 



Contents 



PAGE 

Preface to Thirteenth Edition iii 

Preface to First Edition iv 

List of Illustrations ix 

Collaborative Study Procedures of the Association of 

Official Analytical Chemists xii 

Definitions of Terms and Explanatory Notes xv 

CHAPTER 

1. Agricultural Liming Materials 1 

Calcium Silicate Slags 2 

Elemental Analysis 3 

2. Fertilizers . . . . 7 

Free Water 9 

Phosphorus 9 

Nitrogen 14 

Potassium 18 

Other Elements 20 

Peat 27 

3. Plants 31 

Metals 31 

Nonmetals 38 

Other Constituents 46 

Pigments 49 

Tobacco 51 

4. Disinfectants 56 

Phenol Coefficient 56 

Use-Dilution Method 58 

Available Chlorine Germicidal Equivalent 

Concentration 59 

Sporicidal Test 60 

Fungicidal Test , . 61 

Germicidal and Detergent Sanitizers 61 

Germicidal Spray Products 63 

Water Disinfectants for Swimming Pools 64 * 

Tuberculocidal Activity 65 

Bacteriostatic Activity of Laundry Additives 67 

5. Hazardous Substances 69 

Paint 69 

Preparations Containing Fluorides 69 

Preparations Containing Methanol 70 

Preparations Containing Phenol 71 

Soda Lye 71 

Earthenware 71 

6. Pesticide Formulations 72 

General Methods 72 

Arsenic 72 

Lead 74 

Copper 74 

Zinc 75 

Fluorine 75 



PAGE 

Contamination by Organochlorine Pesticides . . 77 
Volatility of Ester Forms of Hormone-Type 

Herbicides 77 

Inorganic and Organometallic Pesticides and 

Adjuvants 78 

Paris Green 78 

Lead Arsenate 78 

Calcium Arsenate 79 

Zinc Arsenite 79 

Copper Carbonate 79 

Copper Naphthenate 79 

Bordeaux Mixtures 80 

Potassium Cyanate 81 

Cyanides 81 

Lime Sulfur Preparations 81 

Hypochlorites 82 

Chloramine T 83 

Mineral Oils . . ■ 83 

Soap 83 

Mineral Oil-Soap Emulsions 83 

Mercurial Seed Disinfectants 84 

Ant Poisons and Rodenticides 85 

Fumigants 85 

Pesticides Related to Natural Products and Their 

Synergists 86 

Allethrin 86 

Derris and Cube Powder 87 

Pyrethrins 89 

Tobacco and Tobacco Products 90 

Halogenated Pesticides 91 

Aldrin, Dieldrin, and Endrin 91 

Amiben 93 

BHC 93 

Balan or Trifluralin 95 

Captan 96 

Chlordane 97 

Chlorotoluron, Chloroxuron, or Metoxuron .... 99 

Dichlobenil 100 

DDT 100 

Dacthal 102 

Dicamba 1 02 

2,4-D 103 

Dalapon 104 

Dicofol 104 

Fluometuron 105 

Folpet 105 

Heptachlor 1 05 

Picloram-2,4-D 106 

Sodium Trichloroacetate 107 

Nonhalogenated Pesticides 1 07 

Aldicarb 107 

Amitrole 1 07 

Carbaryl 108 

DDVP 108 

Diquat and Paraquat 109 



PAGE 



PAGE 



Dithiocarbamates 110 

Thiram 111 

Dodine 111 

Formaldehyde 111 

Ethion 112 

Formothion 113 

Malathion 113 

Parathion 116 

Phorate 119 

Sulfoxide 120 

Tetraethylpyrophosphate 120 

Organic Thiocyanates 1 20 

Thiocarbamates 121 

Triazines, Diazinon, and Dichiorobenzilates 121 

Triphenyltin Compounds 122 

Quaternary Ammonium Compounds 123 

7. Animal Feed 1 25 

Nitrogen 125 

Other Constituents 132 

Preservatives 141 

8. Baking Powders and Baking Chemicals . . 143 

9. Beverages: Distilled Liquors 147 

Spirits 147 

Cordials and Liqueurs 159 

10. Beverages: Malt Beverages and Brewing Materials 162 

Beer 162 

Malt 171 

Cereal Adjuncts 1 77 

Hops 178 

Brewing Sugars and Sirups 180 

Wort 181 

Yeast 182 

Brewers' Grains 183 

1 1 . Beverages: Wines 1 85 

Preservatives 1 92 

Flavors 193 

12. Beverages: Nonalcoholic and Concentrates 194 

13. Cacao Bean and Its Products 199 

Shell 200 

Cacao Products Processed with Alkalies 205 

Chocolate Liquor 205 

Fat 205 

Dairy Ingredient Constituents 206 

Saccharine Ingredients Other Than Lactose 207 

Starch 208 

Chocolate Products 209 

Theobromine 209 

14. Cereal Foods 211 

Wheat Ftour 211 

Wheat, Rye, Oats, Corn, Buckwheat, Rice, and 
Barley and Their Products Except Cereal 

Adjuncts 220 

Soybean Flour 223 

Bread 223 

Baked Products 226 



Baked Products Other Than Bread (Not 

Containing Fruit) 227 

Fig Bars and Raisin-Filled Crackers 227 

Macaroni, Egg Noodles, and Similar Products . . . 228 

15. Coffee and Tea 233 

Green Coffee 233 

Roasted Coffee 233 

Tea 235 

16. Dairy Products 238 

Sampling 238 

Milk 240 

Automated Methods 246 

Cryoscopic Methods 249 

Penicillins in Milk 255 

Cream 256 

Evaporated Milk 258 

Sweetened Condensed Milk 259 

Dried Milk, Nonfat Dry Milk, and Malted Milk ... 259 

Butter 260 

Cheese 264 

Ice Cream and Frozen Desserts 272 

17. Eggs and Egg Products 275 

18. Fish and Other Marine Products 285 

19. Flavors 306 

Alcohol 306 

Vanilla Extract and Its Substitutes 306 

Lemon, Orange, and Lime Extracts and Flavors . 313 

Lemon and Orange Oils 316 

Almond Extract 318 

Cassia, Cinnamon, and Clove Extracts 319 

Ginger Extract 319 

Peppermint, Spearmint, and Wintergreen 

Extracts 319 

Anise and Nutmeg Extracts 319 

Other Extracts and Toilet Preparations 319 

20. Food Additives: Direct 323 

Acidulants , 323 

Antioxidants 323 

Chemical Preservatives 325 

Emulsifying Agents 343 

Enzymes 344 

Miscellaneous 345 

Nonnutritive Sweeteners 347 

Nutrients 352 

Solvents 352 

21. Food Additives: Indirect 354 

22. Fruits and Fruit Products 359 

Moisture 361 

Solids 362 

Potassium 363 

Sodium 364 

Phosphorus 364 

Betaine 365 

Fruit Acids 366 

Sugars 369 

Anthocyanins 370 



PAGE 

Lemon Juice 372 

23. Gelatin, Dessert Preparations, and Mixes 374 

24. Meat and Meat Products 376 

Meat 376 

Meat Extracts and Similar Products 383 

25. Metals and Other Elements as Residues in Foods . . 385 

Antimony 385 

Arsenic 385 

Cadmium 388 

Copper 391 

Fluorine 392 

Lead 397 

Manganese 405 

Mercury 405 

Nickel 409 

Selenium 409 

Tin 410 

Titanium 411 

Zinc 411 

26. Natural Poisons 414 

Mycotoxins 414 

Aflatoxins 41 5 

Minicolumn Detection Methods 417 

Quantitative Methods 418 

Aflatoxin M, . . . 427 

Ochratoxins 428 

Patulin '. 430 

Sterigmatocystin 431 

Zearalenone 432 

Marine Toxins 433 

Phytotoxins 433 

27. Nuts and Nut Products 435 

Peanut Butter 435 

Shredded Coconut 436 

28. Oils and Fats 437 

. . physical Constants 437 

Chemical Constants 438 

Acid Constituents 441 

Sterols 452 

Tests for Specific Oils 456 

Chick Edema Factor 459 

Mono- and Diglycerides 461 

flaxseed 464 

29. Pesticide Residues 466 

Multiresidue Methods 466 

General Method for Organochlorine and 

Organophosphorus Pesticides 466 

Multiple Residue Methods for 

Organophosphorus Pesticides 476 

Multiple Residue Methods for Fumigants 480 

Multiple Residue Method for Aryl N- 

Methylcarbamate Insecticides 480 

Methods for Individual Residues 481 

Benzene Hexachloride 481 

Biphenyl 481 

Aramite 483 

Captan 483 



PAGE 

Carbaryl 483 

Sulphenone 485 

Perthane 485 

DDT 485 

Dichlone 487 

Guthion 488 

Dodine 488 

Ethylenethiourea 488 

Glyodin 489 

Hexachlorobenzene and Mirex 490 

Malathion 490 

Maleic Hydrazide 490 

Methoxychlor 491 

Mirex 492 

Monofluoroacetic Acid 492 

Naphthaleneacetic Acid 493 

Nicotine 494 

Parathion 495 

Piperonyl Butoxide 495 

Thiram 495 

30. Spices and Other Condiments 497 

Spices 497 

Prepared Mustard 500 

Dressings for Foods 501 

Vinegars 503 

31. Sugars and Sugar Products 506 

Sugars and Sirups 506 

Molasses and Molasses Products 516 

Confectionery 519 

Honey 520 

Maple Products 528 

Sugar Beets 532 

Starch Conversion Products 532 

32. Vegetable Products, Processed 537 

Canned Products 537 

Dried Vegetables 542 

Frozen Vegetables 543 

33. Waters; and Salt 547 

Water 547 

Salt 565 

34. Color Additives 568 

Separation and Identification of Color Additives 

in Foods, Drug?, and Cosmetics 568 

Analysis of >,rcial Synthetic Organic Color 

Additives ?..'.*. . 570 

35. Cosmetics 582 

General Methods 582 

Deodorants and Anti-Perspirants 583 

Dipilatories 587 

Face Powders 587 

Hair Preparations 587 

Suntan Preparations 589 

Vanishing Cream 589 

Bioassay 590 

36. Drugs: General 591 

General Directions 591 

Solvents 591 

Halogenated Compounds 592 



PAGE 

Inorganic Drugs 594 

Mydriatics and Myotics 604 

Microscopic Tests 604 

37. Drugs: Acidic 614 

Acids 614 

Antipyretic Drugs 615 

Hypnotic Drugs 619 

Phenolic Drugs 625 

Sulfonamide Drugs 629 

Other Imide Drugs 631 

38. Drugs: Alkaloid and Related Bases 636 

Alkaloids, Opium 636 

Alkaloids Other Than Opium 639 

Other Bases 649 

39. Drugs: Neutral 667 

Steroids and Related Hormones 667 

Nonalkaloidal Vegetable Drugs and Their 

Derivatives 674 

Miscellaneous 678 

40. Drugs: Illicit 685 

41. Drugs and Feed Additives in Animal Tissues 688 

ANOT 688 

Arsenic 688 

Clopidol 689 

Decoquinate 690 

Ethoxyquin 691 

Melengestrol Acetate 691 

Nalidixic Acid 693 

Zoalene 694 

42. Drugs in Feeds 695 

Qualitative Tests 695 

Arsenic 695 

Cyzine 696 

Aklomide 697 

p-Aminobenzoic Acid 697 

Enheptin 698 

Amprolium 698 

Arsanilic Acid 699 

Bithionol 699 

Buquinolate 699 

Cadmium Anthranilate ■ 700 

Carbadox 700 

Decoquinate 700 

Dibutyltin Dilaurate 701 

Diethylstilbestrol 702 



Nitrophenide 

Nitarsone 

Phenothiazine 

Piperazine 

Pyrantel Tartrate 

Racephenicol 

Reserpine . . . 

Ronnel 

Roxarsone 

Sulfonamide Drugs . . 

Thiabendazole 

Zoalene 

Antibiotics 

43. Vitamins and Other Nutri 

Chemical Methods 

Microbiological Metho 

Bioassay Methods 

Nutritionally Related C 

44. Extraneous Materials: Isc 

General 

Beverages and Bevera 

Dairy Products 

Nuts and Nut Products 
Grains and Their Prodi 

Baked Goods 

Breakfast Cereals 

Eggs and Egg Product; 
Poultry, Meat, and Fisl 

Products 

Fruits and Fruit Produc 
Sugars and Sugar Proi 
Vegetables and Vegetc 
Spices and Other Cone 
Animal Excretions . . . 
Miscellaneous 

45. Forensic Sciences 

Latent Fingerprints . . 

Glass Fragments 

Mineral Wool Insulatic 
Voice Print Identificatic 

46. Microbiological Methods 

Eggs and Egg Product 
Frozen, Chilled, Precoc 
Commercial Sterility o 
Thermophilic Bacterial 
Clostridium perfringen 
Salmonella .......... 

Optical Somatic Cell C 



Illustrations 



FIGURE PAGE 

1 .01. Apparatus for Automatic Filtration and Measurement of Lime Solutions 2 

2:01. Sampling Pattern 7 

2:02. Sampling Cup 7 

2:03. Missouri and Indiana Weighted Restricted-fill Fluid Fertilizer Sampling Bottles Designed to Fill While Being Lowered 

(and Raised) in Storage Tanks 7 

2:04. Sampling Apparatus for Ammoniacal Solutions, Including "Quick Coupler" for Attaching to Storage Tanks 8 

2:05. Apparatus for Control of Water Pressure 9 

2:06. Flow Diagram for Automated Analysis for Phosphorus 12 

2 :07. Flow Schematic for K 2 in Fertilizers 20 

2:08. Apparatus for Elution of Resin Column 26 

3:01 . Suction Device Used in Micro Method for Determining Calcium 32 

3:02. Schematic Drawing of Air Flow System Used in Semiautomated Analysis for Fluoride 41 

3:03. Flow Diagram for Semiautomated Analysis for Fluoride 42 

3:04. Schematic Drawing of Microdistillation Apparatus 43 

3:05. Microdistillation Column 43 

3:06. Apparatus for Determining Lignin 48 

3:07. Distillation Apparatus 54 

4:01 . Transfer Loop and Manner of Using in Phenol Coefficient Technic : 57 

4:02. Stainless Steel Spindle for Winding Test Fabric 67 

6:01 . Delivery Assembly for Sampling Pressurized Containers 72 

6:02. Apparatus for Distilling Arsenious Chloride 73 

6:03. Apparatus for Determining Fluorine 76 

6:04. Partition Column and Solvent Evaporator 94 

6:05. Melting Point Apparatus 94 

6:06. Distillation Apparatus 99 

6:07. Carbon Disulfide Evolution Apparatus i\ 110 

6:08. Absorption System for Thiram . 111 

6:09. Filtration Apparatus 122 

7:01. Flow Diagram for Semiautomated Analysis for Crude Protein 128 

7:02. Agitator 131 

7:03. Oklahoma State Filter Screen 133 

7:04. Modified California State Buchner Funnel 133 

7:05. Continuous Heater for Distilled Water, 1.25% Alkali, and 1.25% Acid ., 133 

8:01. Chittick Apparatus for Gasometric Determination of Carbon Dioxide > 143 

9:01. 100 mL and 50 mL Pycnometers 148 

9:02. Williams Tube 149 

9:03. Steam Distillation Flask 156 

9:04. Apparatus for Determining Hydrogen Cyanide in Distilled Spirits 158 

10:01. Absorption Buret 166 

11:01. Distillation Apparatus for Chemical Determination of Alcohol in Wine 186 

1 1 :02. Volatile Acid Still (Cash Still) 188 

1 1 :03. Carbon Dioxide Apparatus; Manometric Method 190 

1 1 :04. Carbon Dioxide Apparatus; Volumetric Method 192 

1 2:01 . Continuous Extraction Apparatus 195 

13:01, Spiral Vessel Sections 203 

13:02. Stone Cells 204 

13:03. Graph Used in Correcting Cuprous Oxide for Effect of Sucrose 207 

1 6:01 . Apparatus for Drying Pentabromacetone by Aspiration 241 

16:02. Liquid Extractor 242 

16:03. Flow Diagram for Determination of Fat in Milk 247 

1 6:04. Modified Jones Reductor 266 

16:05. Modified Plastic Desiccator 273 

18:01. Extraction Apparatus 291 

1 8:02. Steam Distillation Assembly 292 

ix 



FIGURE PAGE 

18:03. Muscle Bath 294 

18:04. Electrophoresis Cabinet 300 

18:05. Disc Electrophoresis Apparatus 300 

18:06. Key to Identification of Canned Salmon Species by Scale Characteristics 302 

18:07. Sockeye (Red) Salmon Scale (Oncorhynchus nerka) 303 

18:08. Chinook (King) Salmon Scale {Oncorhynchus tshawytscha) 304 

18:09. Coho (Silver) Salmon Scale {Oncorhynchus kisutch) 304 

18:10. Chum Salmon Scale {Oncorhynchus keta) 304 

18:1 1. Pink Salmon Scale {Oncorhynchus gorbuscha) 304 

19:01. Wilson Flask 315 

19:02. Oil Separator Trap 320 

20:01. Steam Distillation Apparatus 326 

20:02. Placement of Slides on Template for Applying Silica Gel Coating , . 327 

20:03. Steam Distillation Apparatus 338 

20:04. Apparatus for Modified Monier-Williams Method for Sulfur Dioxide 339 

20:05. Alternative S0 2 Absorber 339 

20:06. Gas Chromatograms of TBS, DBS, and TBS + DBS 346 

20:07. Microsampling Die 348 

20:08. GLC Effluent Collection Trap for Determination of Cyclohexylamine 350 

21 :01. Test Cell 356 

21:02. Oven Rack 357 

22:01. Apparatus for Determining Volume of Frozen Fruits by Displacement 359 

22:02. Capillary Viscometer 361 

22:03. Electrical Circuit Diagram for Dried Fruit Moisture Tester 361 

22:04. Connecting Tube Adapter for Direct Distillation 370 

23:01. 0.5 Inch Bloom Gelometer Plunger , 374 

23:02. 1.0 Inch Bloom Gelometer Plunger 375 

24:01. Helix Inlet Manifold 377 

24:02. Phosphorus Analytical Manifold 378 

24:03. Nitrogen Analytical Manifold 379 

25:01 . Arsenic Apparatus 386 

25:02. Apparatus for Flameless Atomic Absorption Analysis 406 

25:03. Digestion Vessel 407 

25:04. Special Digestion Apparatus for Mercury Residues 408 

26:01 . Spotting and Scoring Patterns for 2-Dimensional TLC Plates 424 

26:02. Plate Scraper for Removing Adsorbent From TLC Plates 424 

26:03. Schematic Showing Parts of Egg and Modes of Test Material Introduction 426 

28:01. Titer Stirring Assembly 439 

28:02. Liquid-Liquid Extractor 442 

28:03. Apparatus for Determining Reichert-Meissl and Polenske Values 443 

28:04. Constant Temperature Bath and Accessories 444 

28:05. Distribution Heads; Manifold 445 

28:06. Glass Micro Filter for Sterol Acetate Precipitates 452 

28:07. Crystalline Forms of Free Sterols 453 

28:08. Apparatus for Determining Melting Point - 458 

29:01 . KCI Thermionic Detector Coil for In-series Dual Detection System 468 

29:02. KCI Thermionic Detector Coil for Parallel and In-series Split Dual Detection Systems 469 

29:03. In-series Split Dual Detection System 469 

29:04. Parallel Dual Detection System 469 

29:05. In-series Dual Detection System 469 

29:06. Sweep Co-distillation Apparatus 477 

29:07. Evaporative Concentrator 484 

29:08. Distillation Apparatus for Maleic Hydrazide Determination 491 

30:01. Apparatus for Volatile Oil in Spices 498 

31 :01 . Fractionator 506 

31 :02. Carbon Combustion and Purification System 527 

31 :03. Distillation Apparatus for Determining Formaldehyde in Maple Sirup 530 

33:01. Apparatus for Determination of Mercury by Flameless Atomic Absorption 560 

33:02. Phosphorus Manifold 563 

34:01 . Titanous Chloride Titration Apparatus 574 

34:02. Sulfiding Apparatus 579 

36:01 . Apparatus for Determining Arsenic in Iron-Arsenic Tablets 594 

36:02. Flow Diagrams for Semiautomated Analysis for Ferrous Sulfate 601 

37:01. Bromine Apparatus 619 



FIGURE PAGE 

37:02. Flow Diagram for Automated Analysis for Acenocoumarol, Dicumarol, Phenprocoumon, Potassium Warfarin, and 

Sodium Warfarin . . 629 

38:01 . Flow Diagram for Semiautomated Fluorometric Analysis for Reserpine 646 

38:02. Flow Diagram for Phenylephrine Hydrochloride 661 

38:03. Assembly of Debubbler 662 

39:01 . Flow Diagram for Semiautomated Analysis for Prednisolone or Prednisone 674 

39:02. Flow Diagram for Automated Analysts for Digoxin 675 

39:03. Scrubber Trap for Ammonia Distillation 679 

39:04. Flow Diagram for Automated Analysis of Methenamine and Methenamine Mandelate 683 

43:01. Flow Diagram for Automated Analysis for Niacin and Niacinamide 745 

43:02. fl/?/?-a-Tocopherol 752 

43:03. Line Test Chart 771 

43:04. Radii Sections Scored According to Line Test Chart 772 

44:01. Mechanical Butter Stirrer 781 

44:02. Wildman Trap Flask 781 

44:03. Rot Fragment Counting Slide 782 

44:04. Sediment Filtering Apparatus, Unassembled 786 

44:05. Sediment Filtering Apparatus, Assembled 786 

44:06. Rot Fragments from Tomato Puree 800 

44:07. Rot Fragments from Tomato Puree 801 

44:08. Mold Filaments in Tomato Products .....' 802 

44:09. Insect Penetration of Packaging 817 

46:01 . Plastic Template Schematic for Microslide Assembly 842 

46:02. Arrangement of Antisera and Homologous Reference Enterotoxins 843 

46:03. Examples of 4 Possible Reactions in Bivalent Detection System 844 

46:04. Effect of Amount of Enterotoxin in Test Preparation on Development of Reference Line of Precipitation 845 

46:05. Appearance of Microslide Gel Diffusion Test as Monovalent System 845 

46:06. Precipitate Patterns in Microslide Gel Diffusion Test Demonstrating Nonspecific (Atypical) Lines of Precipitation 845 

46:07. Optical Somatic Cell Counter Flow Diagram 846 

46:08. Optical Somatic Cell Counter Flow Diagram 848 

46:09. Optical Somatic Cell Counter Flow Diagram 849 

47:01. Combustion Tube 853 

47:02. Carbon and Hydrogen Apparatus 855 

47:03. Upper Section of Distilling Apparatus 857 

47:04. Gravimetric Setup for Oxygen Determination 859 

47:05. Quartz Reaction Tube and Filling for Oxygen Determination 859 

47:06. Details of Modified Clark Apparatus 862 

47:07. Modified Clark Apparatus 863 

48:01 . Cross-section of Marinelli Beaker 869 



Collaborative Study Procedures of the Association of 

Official Analytical Chemists 



The Association of Official Analytical Chemists (AOAC) is a 
unique, nonprofit scientific organization whose primary purpose 
is to serve the needs of government regulatory and research 
agencies for analytical methods. The goal of the Association is 
to provide methods which will perform with the necessary 
accuracy and precision under usual laboratory conditions (/). 
Since its formation in 1 884 the AOAC has provided a mechanism 
to select methods of analysis from published literature or 
develop new methods, collaboratively test them through inter- 
laboratory studies, approve them, and publish the approved 
methods for a wide variety of materials relating to foods, drugs, 
cosmetics, agriculture, forensic science, and products affecting 
the public health and welfare. Its membership is composed of 
scientists from Federal, State, Provincial, and other regulatory 
bodies who work within the AOAC's established procedures as 
researchers, methods collaborators, and committee members. 
Although most of the members are from North America, many 
nations throughout the world are represented. 

The AOAC has almost a century of experience in utilizing the 
collaborative study as a means of determining the reliability of 
analytical methods for general purposes and, especially, for 
regulatory purposes. In fact, the AOAC's major contribution to 
analytical science has been to bring the collaborative study 
technique for the validation of analytical methods to a high 
degree of perfection. In such a study, laboratories analyze 
identical sample sets which cover the range of applicability of 
a method previously selected as being useful and practical. The 
purpose of the study is to establish the characteristics of the 
methods with respect to accuracy, precision, sensitivity, range, 
specificity, limit of detection, limit of reliable measurement, 
selectivity, practicality, and similar attributes, as required. 

ORGANIZATION AND PROCEDURES FOR AOAC 
COLLABORATIVE STUDIES 

The collaborative study is organized and directed by an analyst 
designated as the Associate Referee for the specific subject 
under investigation. Currently, some 600 Associate Referees 
appointed by the Association are responsible for as many topics. 
An Associate Referee is selected for his knowledge, interest, 
and experience in the subject matter field. He operates under 
the scientific guidance, support, and administrative supervision 
of a General Referee, who is in turn responsible for a product 
area. The Associate Referee reviews the literature and selects 
one or two of the better analytical methods available, modifying 
them as needed. Alternatively, he may develop or adapt a 
method used in his laboratory for the analyte and matrix under 
study, testing it thoroughly in his laboratory before designing 
a collaborative study. The General Referee is kept informed of 
such preliminary studies. 

The samples analyzed in a collaborative study are normally 
prepared and distributed to the participants by the Associate 
Referee. The Association follows the recommendations of You- 
den (2) that not fewer than five laboratories participate and that 
a minimum of six sample materials be sent to each. These are 



minima and, in practice, both are usually exceeded. In addition, 
a reference or practice sample is included, where possible. 

Laboratories with at least some experience in the general 
subject matter are selected as collaborators. Because the objec- 
tive of the study is to standardize the method, as contrasted to 
standardizing the analyst {3), all analysts are instructed to follow 
the method exactly as written even though they may not concur 
with the Associate Referee's selection among possible alterna- 
tives. The level of the analyte in the samples is usually unknown 
to the participants. 

All individual results obtained by the collaborators are re- 
ported to the Associate Referee, who compiles and evaluates 
them. Since statistical treatment of the data is considered 
essential in a rigorous evaluation of the method for accuracy, 
precision, sensitivity, and specificity, it is now required for all 
studies. The Association considers this of such importance that 
it provides statistical assistance in all cases where it is otherwise 
unavailable to the Associate Referee. A statistical manual {4) is 
also provided. 

The Associate Referee makes the initial judgment on the 
performance of the method. If he recommends approval, it 
passes to the General Referee and then to a committee of 
experts. If both recommend approval, the method is presented 
at the Association's annual business meeting for vote by the 
membership. 

Approved methods and supporting data are published in the 
Journal of the Association of Official Analytical Chemists. They 
are subject to scrutiny and general testing by other analysts for 
at least a year before final adoption. They may be modified and 
restudied collaboratively as needed, should feedback from gen- 
eral use reveal flaws in the method or in its written set of 
directions. Approved methods are included in the Association's 
"Official Methods of Analysis", a book of some 1000 pages 
which is updated every 4-5 years. 

The preceding summary of AOAC's modus operandi recog- 
nizes the need for healthy skepticism toward results obtained 
by analytical methods which have not undergone such rigorous 
scrutiny and interlaboratory testing of their accuracy, precision, 
dependability, specificity, and practicality. 

SELECTION OF METHODS FOR STUDY 

A certain degree of variability is associated with all measure- 
ments. Much of the research on analytical chemistry is an 
attempt to minimize that variability. But there are many different 
types of variability in analytical work. We often find that when 
we attempt to minimize one kind, we must necessarily permit 
expansion in another kind. In practical analytical chemistry, the 
problem often comes down to which variability is to be mini- 
mized. 

Some examples of this point may be helpful. In atomic weight 
determination, everything — espec\a\\y practicality — is sacrificed 
for accuracy. A high degree of accuracy and practicality is 
required in the assay of precious metals, but the fire assay used 
is generally applicable to little else besides metals and minerals. 



In clinical chemistry, within-laboratory precision (repeatability) 
is critical, and often is of greater interest to clinical laboratories 
than absolute accuracy or agreement with the values of other 
laboratories (reproducibility). In drug analysis, a high degree of 
accuracy is required in the therapeutic range because the 
analytical values determining the identity, strength, quality, and 
purity of pharmaceutical preparations, as laid down in phar- 
macopoeial specifications, are directly related to clinical value. 
With polynu clear hydrocarbons, specificity is important, since 
some of these compounds are carcinogenic while others are 
not. In applying the famous Delaney clause of the United States 
Federal Food, Drug, and Cosmetic Act, all attributes of the 
analytical methods are secondary to the detection of extremely 
small concentrations (detectability), or to exhibiting a high 
degree of response for small changes in concentration {sensi- 
tivity). 

There is a very special case involving accuracy, where the 
"true value" is determined by the method of analysis. Many 
legal specifications and standards for food and agricultural 
products define ill-defined components such as moisture, fat, 
protein, and crude fiber in terms of reference methods. There- 
fore, the precision of these methods becomes the limiting factor 
for their performance. In fact, most analyses involved in com- 
mercial transactions require primarily that the buyer and seller 
agree on the same value (analytically and economically), re- 
gardless of where it stands on an absolute scale. 

The point of these examples is that although methods of 
analysis are characterized by a number of attributes — accuracy, 
precision, specificity, sensitivity, detectability, dependability, 
and practicality — no method is so flawless that all these qualities 
can be maximized simultaneously. For any particular analysis, 
the analyst must determine, on the basis of the purpose of the 
analysis, which attributes are essential and which may be 
compromised. 

Unfortunately, the literature is replete with examples indicat- 
ing that an individual analyst, and especially the originator of 
a method of analysis, is not an unbiased judge of the relative 
merits of the methods of analysis which he develops and uses. 
In our experience, the collaborative study provides impartial 
data on the suitability of the method. The data, in many cases, 
speak for themselves. 

The collaborative study, or ring test or round robin test, as it 
is called in other organizations, provides the basic information 
on the performance of analytical methods. The extent of the 
information will depend on the number of samples provided, 
the number of analyses performed, and the number of labora- 
tories participating. The data should be unbiased because the 
composition of the samples is known only to the administrator 
of the study. Some of the requirements of the study and their 
relationship to the characteristics and attributes of the method 
are as follows: 

(1) Accuracy. Samples must be of defined composition (by 
spiking, by formulation, or by analytical consensus). 

(2) Specificity. Samples should contain related analytes. 

(3) Sensitivity. Samples should differ from each other or from 
negative samples by a known amount. 

(4) Applicability. Samples should include the concentration 
range and matrix components of interest. 

(5) Blanks. Samples should include different matrices with 
"none" of the component of interest. 

(6) Precision. Instructions should request replicate analyses 
by the same or different analysts in the same laboratory, 
preferably on different days. By far a better procedure is to 
include "blind" (unknown to the analyst) replicate samples in 
the series. 

(7) Practicality. Instructions should request information as to 



the actual and elapsed time required for the analyses; the 
availability of reagents, equipment, and standards; and any 
necessary substitutions. When practice samples are included, 
the number of analyses required to achieve the stated recovery 
and repeatability, should be reported. 

PROCEDURAL DETAILS OF COLLABORATIVE STUDY 

As numerous beginners in this field have discovered, much 
preliminary work must be done before sending out samples: 

(1) The method must be chosen and demonstrated to apply 
to the matrices and concentrations of interest. 

(2) The critical variables in the method should have been 
determined and the need for their control emphasized [a rug- 
gedness test (5) is useful for this purpose]. 

(3) The method should be written in detail by the Associate 
Referee and tested by an analyst not previously connected with 
its development. 

(4) Unusual standards, reagents, and equipment must be 
available from usual commercial sources of supply, or sufficient 
quantities must be prepared or obtained to furnish to the 
participants. 

(5) The samples must be identical and homogeneous so that 
the analytical sample error is only a negligible fraction of the 
expected analytical error. 

(6) A sufficient number of samples must be prepared to cover 
typical matrices and the concentration range of interest (toler- 
ance, maximum or minimum specifications, likely levels of 
occurrence, etc.). 

(7) Samples must be stable and capable of surviving the 
rigors of commercial transportation. 

(8) Reserve samples should be prepared and preserved to 
replace lost samples and to permit reanalysis of samples con- 
sidered as outliers to attempt to discover the cause of abnormal 
results. 

(9) The instructions must be clear. They should be reviewed 
by someone not connected with the study to uncover potential 
misunderstandings and ambiguities. 

(10) If the analyte is subject to change (e.g., bacterial levels, 
nitroglycerin tablets), provision must be made for all participants 
to begin the analysis at the same time. 

(11) Practice samples of a known and declared composition 
should be furnished with instructions not to analyze the un- 
knowns until a specified degree of recovery and repeatability 
(or other attribute) has been achieved. 

(12) Provision should be made when necessary for submis- 
sion of standard curves, tracings of recorder charts, or photo- 
graphs of thin-layer plates in order to assist in determining 
possible causes of error. 

OTHER TYPES OF BNTERLABORATORY STUDIES 

This type of collaborative study, which is designed to deter- 
mine the characteristics of a method, must be carefully distin- 
guished from other types of interlaboratory studies which by 
design or through ignorance provide other kinds of information. 
The most important types of other studies are: 

(1) Those studies which require the collaborators to investi- 
gate the variability of parts of methods or applicability to 
different types of samples. (An interlaboratory study is usually 
an inefficient way of obtaining this type of information.) 

(2) Those studies which permit an analyst to use any method 
he desires. Such studies invariably produce such a wide scatter 
of results that the data are of little value for evaluation of 
methods. They may be useful in selecting a method from a 
number of apparently equivalent methods, provided the purpose 



is emphasized beforehand and the participants provide a de- 
scription of the method used in order to permit a correlation of 
the details of the methods with apparent biases and variabilities. 

(3) Those studies which are used for quality control purposes, 
whose participants are not permitted sufficient time to gain 
familiarity with the method, or who permit deviations to enter 
into the performance of the analyses on the grounds that the 
deviation is obviously an improvement which could not possibly 
affect the results of the analysis, or who claim to have a superior 
method. 

With this background information, it is now appropriate to 
introduce the following definitions which were agreed upon as 
part of the guidelines for collaboration between the AOAC and 
the Collaborative International Pesticide Analytical Council Ltd. 
(CIPAC) (6). 

Collaborative study. An analytical study involving a number 
of laboratories analyzing the same sample(s) by the same 
method(s) for the purpose of validating the performance of the 
method(s). 

Preliminary interlaboratory study. An analytical study in which 
two or more laboratories evaluate a method to determine if it 
is ready for a collaborative study. 

Laboratory performance check. The analysis of very carefully 
prepared and homogeneous samples, normally of known active 
ingredient content, to establish or verify the performance of a 
laboratory or analyst. 

SUMMARY 

The collaborative study is an experiment designed to evaluate 
the performance of a method of analysis through the analysis 
of a number of identical samples by a number of different 



laboratories. With proper design, it provides an unbiased eval- 
uation of the performance of a method in the hands of those 
analysts who will use it. A collaborative study must be distin- 
guished from those studies designed to choose a method or to 
determine laboratory or analyst performance. 

REFERENCES 

(1) AOAC, "Handbook of the AOAC", 4th ed., AOAC, Box 540, 
Benjamin Franklin Station, Washington, D.C. 20044, 1977. 

(2) W. J. Youden, "Accuracy of Analytical Procedures", J. Assoc. 
Off. Anal. Chem., 45, 169-73 (1962). 

(3) Harold Egan, "Methods of Analysis; An Analysis of Meth- 
ods", ibid., 60, 260-7 (1977). 

{4) W. J. Youden and E. H. Steiner, "Statistical Manual of the 
AOAC: Statistical Techniques for Collaborative Tests. Plan- 
ning and Analysis of Results of Collaborative Tests", AOAC, 
Box 540, Benjamin Franklin Station, Washington, D.C. 20044, 
1975. 

(5) W. J. You den /The Collaborative Test", J. Assoc. Off. Anal. 
Chem. f 46, 55-62 (1963). 

{6) "Guidelines for Collaboration Between the Association of 
Official Analytical Chemists (AOAC) and the Collaborative 
International Pesticide Analytical Council Ltd. (CIPAC)", ibid., 
57,447-9(1974). 

BIBLIOGRAPHY 

Daniel Banes, "The Collaborative Study as a Scientific Concept", 
J. Assoc. Off. Anal. Chem., 52, 203-06 (1969). 

William Horwitz, "Problems of Sampling and Analytical Meth- 
ods", ibid, 59, 1197-203 (1976). 



Reprinted with permission from: Analytical Chemistry (March 1978) 50, 337A-340A. 
Published 1978 American Chemical Society 



Definitions of Terms and Explanatory Notes 



Reagents 

(1 ) Term "H 2 0" means distilled water, except where otherwise 
specified, and except where the water does not mix with the 
detn, as in "H 2 bath/' 

(2) Term "alcohol" means 95% ethanol by vol. Alcohol of 
strength x% may be prepd by dilgx mL 95% alcohol to 95 mL 
with H 2 0. Absolute alcohol is 99.5% by vol. Formulae of specially 
denatured alcohols (SDA) used as reagents are as follows: 

SDA No. 100 parts alcohol plus 

1 5 wood alcohol 

2-B 0.5 benzene or rubber 

hydrocarbon solv. 
3-A 5 MeOH 

12-A 5 benzene 

13-A 10 ether 

23-A 10 acetone 

30 10 MeOH 

"Reagent" alcohol is 95 parts SDA 3-A plus 5 parts isopropanol. 

(3) Term "ether" means ethyl ether, peroxide-free by follow- 
ing test: To 420 mL ether in separator add 9.0 mL 1% NH 4 V0 3 
in H 2 S0 4 (1+16). Shake 3 min and let sep. Drain lower layer into 
25 mL g-s graduate, dil. to 10 mL with H 2 S0 4 (1+16), and mix. 
Any orange color should not exceed that produced by 0.30 mg 
H 2 2 (1 mL of soln prepd by dilg 1 mL 30% H 2 2 to 100 mL with 
H 2 0) and9.0mL1% NH 4 V0 3 in H 2 S0 4 (1 + 16). Peroxides may be 
eliminated by passing <700 mL ether thru 10 cm column of 
Woelm basic alumina in 22 mm id tube. 

(4) Reagents listed below, unless otherwise specified, have 
approx. strength stated and conform in purity with Recom- 
mended Specifications for Analytical Reagent Chemicals of 
American Chemical Society: 

Assay 

Sulfuric Acid 95.0-98.0% H 2 S0 4 

Hydrochloric acid 36.5-38.0% HCI 

Nitric acid 69.0-71.0% HN0 3 

Fuming nitric acid s=90% HN0 3 

Acetic acid , 5*99.7% HC 2 H 3 2 

Hydrobromic acid 47.0-49.0% HBr 

Ammonium hydroxide 28-30% NH 3 

Phosphoric acid s=85% H 3 P0 4 

Where no indication of diln is given, reagent is of concn given 
above. 

(5) All other reagents and test solns, unless otherwise de- 
scribed in text, conform to requirements of American Chemical 
Society. Where such specifications have not been prepd, use 
highest grade reagent. When anhyd. salt is intended, it is so 
stated; otherwise the crystd product is meant. 

(6) Unless otherwise specified, phenolphthalein (phthln) used 
as indicator is 1% ale. soln; Me orange is 0.1% aq. soln; Me red 
is 0.1% ale. soln. 

(7) Directions for stdzg reagents are given in Chapter 50. 

(8) Unusual reagents not mentioned in reagent sections or 
cross referenced, other than common reagents normally found 
in laboratory, are italicized first time they occur in method. 

(9) Com. prepd reagentsolns must be checked for applicability 
to specific method. They may contain undeclared buffers, pre- 
servatives, chelating agents, etc. 



(10) In expressions (1+2), (5+4), etc., used in connection with 
name of reagent, first numeral indicates vol. reagent used, and 
second numeral indicates vol. H 2 0. For example, HCI (1+2) 
means reagent prepd by mixing 1 vol. HCI with 2 vols H 2 0. 
When one of reagents is solid, expression means parts by wt, 
first numeral representing solid reagent and second numeral 
H 2 0. Solns for which the solv, is not specified are aq. solns. 

(11) In making up solns of definite percentage, it is understood 
thatx g substance is dissolved in H 2 and dild to 100 mL. Altho 
not theoretically correct, this convention will not result in any 
appreciable error in any of methods given in this book. 

(12) Chromic acid cleaning soln is prepd by (7) adding 1 L 
H 2 S0 4 to ca 35 mL satd aq. Na 2 Cr 2 7 soln; or (2) adding 2220 mL 
(9 lb) H 2 S0 4 to ca 25 mL satd aq. Cr0 3 soln (170 g/100 mL). 
Reagents may be tech. grade. Use only after first cleaning by 
other means (e.g., detergent) and draining. Mixt. is expensive 
and hazardous. Use repeatedly until it is dild or has a greenish 
tinge. Discard carefully with copious amts of H 2 0. 

(13) All calcns are based on table of international atomic 
weights, 52.001. 

Apparatus 

(14) Burets, vol. flasks, and pipets conform to following 
Federal specifications (available from General Services Admin., 
Specification Activity 3F1, Washington Navy Yard, Bldg. 197, 
Washington, DC 20407): 

Buret NNN-B-00789a May 19, 1965 

Flask, vol. NNN-F-00289d Feb 7, 1977 

Pipet, vol. NNN-P-395c March 13, 1970 

Pipet, measuring NNN-P-350c July 16, 1973 

See also NBS Circular 602, "Testing of Glass Volumetric Ap- 
paratus" (available as Com 73-10504 from NTIS, Springfield, VA 
22151). 

(15) Standard taper {$) glass joints may be used instead of 
stoppers where the latter are specified or implied for connecting 
glass app. 

(16) Sieve designations, unless otherwise specified, are those 
described in Federal Specification RR-S-366e, Nov 9, 1973 (avail- 
able from General Services Admin.). Designation " '100-mesh' 
(or other number) powder (material, etc.)" means material 
ground to pass thru std sieve No. 100 (or other number). 
Corresponding international std and US std sieves are given in 
Table 1. 

(17) Term "paper" means filter paper, unless otherwise spec- 
ified. 

(18) Term "high-speed blender" designates mixer with 4 
canted, sharp-edge, stainless steel blades rotating at the bottom 
of 4-lobe jar at 10,000-12,000 rpm, or with equiv. shearing 
action. Suspended solids are reduced to fine pulp by action of 
blades and by lobular container, which swirls suspended solids 
into blades. Waring Blendor, or equiv., meets these require- 
ments. 

(19) "Flat-end rod" is glass rod with one end flattened by 
heating to softening in flame and pressing vertically on flat 
surface to form circular disk with flat bottom at end. 

(20) Designation and pore diam. range of fritted glassware 
are: extra coarse, 170-220 /urn; coarse, 40-60; medium, 10-15; 
fine, 4-5.5; Jena designations and pore diam. are: 1, 110 jum; 
2, 45; 3, 25; 4, 8. 



(21) Unless otherwise indicated, temps are expressed as 
degrees Centigrade. 

Table 1. Nominal Dimensions of Standard Test Sieves (U.S.A. 
Standard Series) 





Sieve Designation 


Nominal 


Nominal 


International 

Standard 3 

(ISO) 


U.S.A. 
Standard 


Sieve 

Opening, 

inches 


Wire 
Diameter, 

mm 


12.5 


mm 1 


V 2 in.* 


0.500 


2.67 


11.2 


mm 


7 /ie in. 


0.438 


2,45 


9.5 
8.0 


mm 

mm 


3 / 8 in. 
5 /ie in. 


0.375 
0.312 


2.27 
2.07 


6.7 
6.3 


mm 
mm" 


0.265 in. 
V 4 in. fi 


0.265 
0.250 


1.87 
1.82 


5.6 


mm 


No. 3V 2 


0.223 


1.68 


4.75 
4.00 


mm 
mm 


No. 4 
No. 5 


0.187 
0.157 


1.54 
1.37 


3.35 
2.80 


mm 
mm 


No. 6 
No. 7 


0.132 
0.111 


1.23 
1.10 


2.38 
2.00 


mm 
mm 


No. 8 
No. 10 


0.0937 
0.0787 


1.00 
0.900 


1.70 


mm 


No. 12 


0.0661 


0.810 


1.40 


mm 


No. 14 


0.0555 


0.725 


1.18 


mm 


No. 16 


0.0469 


0.650 


1.00 


mm 


No. 18 


0.0394 


0.580 


850 


/xm c 


No. 20 


0.0331 


0.510 


710 
600 
500 
425 


jitm 

^m 
/i,m 


No. 26 
No. 30 
No. 35 
No. 40 


0.0278 
0.0234 
0.0197 
0.0165 


0.450 
0.390 
0.340 
0.290 


355 


)w,m 


No. 45 


0.0139 


0.247 


300 
250 
212 
180 
150 
125 


jum 
/xm 
/tm 
/im 
fim 
/Am 


No. 50 
No. 60 
No. 70 
No. 80 
No. 100 
No. 120 


0.0117 
0.0098 
0.0083 
0.0070 
0.0059 
0.0049 


0.215 
0.180 
0.152 
0.131 
0.110 
0.091 


106 
90 


/am 
/Am 


No. 140 
No. 170 


0.0041 

0.0035 


0.076 
0.064 


75 


tim 


No. 200 


0.0029 


0.053 


63 


Aim 


No. 230 


0.0025 


0.044 


53 


/xm 


No. 270 


0.0021 


0.037 



a These standard designations correspond to the values for test sieve 
apertures recommended by the International Organization for Standard- 
ization, Geneva, Switzerland. 

b These sieves are not in the standard series but they have been 
included because they are in common usage. 

c 1000 /Am = 1 mm. 



Standard Operations 

(22) Operations specified as "wash (rinse, ext. etc.) with two 
(three, four, etc.) 10 mL (or other vol.) portions H 2 (or other 
solv.)" mean that the operation is to be performed with indicated 
vol. of solv. and repeated with same vol. of solv. until number 
of portions required have been used. 

(23) Definitions of terms used in methods involving spectro- 
photometry are those given in JAOAC 37, 54(1954). Most 
important principles and definitions are: 

(a) More accurate instrument may be substituted for less 
accurate instrument (e.g., spectrophtr may replace colorimeter) 
where latter is specified in method. Wavelength specified in 
method is understood to be that of max. absorbance [A), unless 
no peak is present. 

(b) Absorbance{s) {A). — Neg. logarithm to base 10 of ratio of 
transmittance (7") of sample to that of ref. or std material. Other 



names that have been used for quantity represented by this 
term are optical density, extinction, and absorbancy. 

(c) Absorptivity {ies) (a). — Absorbance per unit con en and cell 
length, a ~ A/bc, where b is in cm and c in g/L, or a =. {A/bc) 
x 1000, if c is in mg/L. Other names that have been used for 
this or related quantities are extinction coefficient, specific 
absorption, absorbance index, andf 1 ^. 

(d) Transmittanceis) (T).— Ratio of radiant power transmitted 
by sample to radiant power incident on sample, when both are 
measured at same spectral position and with same slit width. 
Beam is understood to be parallel radiation and incident at right 
angles to plane parallel surface of sample. If sample is soin, 
solute transmittance is quantity usually desired and is detd 
directly as ratio of transmittance of soln in cell to transmittance 
of solv. in an equal cell. Other names that have been used for 
this quantity are transmittancy and transmission. 

(e) Standardization. — Spectrophtr may be checked for accu- 
racy of wavelength scale by ref. to Hg lines: 239.95, 248.3, 
253.65, 265.3, 280.4, 302.25, 313.16, 334.15, 365.43, 404.66, 
435.83, 546.07, 578.0, and 1014.0 nm. To check consistency of 
absorbance scale, prep, soln of 0.0400 g K 2 Cr0 4 /L 0.05/V KOH 
and det. absorbance at following wavelengths in 1 cm cell: 230 
nm, 0.171; 275, 0.757; 313.2, 0.043; 375, 0.991; 400, 0.396. See 
"Standards for Checking the Calibration of Spectrophotome- 
ters/' Letter Circular LC-1017, reissued Jan 1967, NBS. 

(24) Least square treatment of data and calculation of regres- 
sion lines, — This technic finds the best fitting straight line for 
set of data such as std curve. It calcs that straight line whose 
sum of squares of vertical deviations (usually>4) of observations 
from the line is smaller than corresponding sum of squares of 
deviations from any other line. Equation of straight line is: 

Y = a + bX, 
where a is intercept at Y axis (X = 0), and b is slope of line. 

Least square estimates of constants are: 

S(x, y,)-[gXiSr,}//»] 



b=- 



IXj - (ZX-y/n 
~bX, 



a = Y 

where X = "sum of^ the n individual values of indicated 
operation, and X and Y are the averages of the X and Y points. 
Example; To find "best" straight line relating A (Y) to concn 
(X): 



Observation 

No. (,) 



Concn 

X, 



Absorbance 
Y, 



X,V, 



1 


80 


1.270 


6400 


101.6 


2 


60 


1.000 


3600 


60.0 


3 


40 


0.700 


1600 


28.0 


4 


30 


0.550 


900 


16.5 


5 


20 


0.250 


400 


5.0 


6 


10 


0.100 


100 


1.0 


7 





0.050 





0.0 



Totals: 

n = 1 



ZX, = 240 1Y; = 3.92 XX? 



13000 X(X,y,) = 212.1 



X 
Y 



■- IXJn 
■ lYJn - 
212.1 - 



= 240/7 - 34.29 
3.92/7 = 0.56 
(240)(3.92)/7 



77.7 



= 0.0163 



13000 ~(240) 2 /7 4771 
a = 0.56 - 0.0163(34.29) = 0.001 
Best equation is then: 

/ = 0.00 + 0.01 63X 
If for sample, A - 0.82, corresponding concn (X) would be: 
X = (Y - 0.00)/0.0163 =0.82/0.0163 - 50.3. 



Many scientific and statistical calculators are preprogrammed 
to perform this calcn. 

(25) Common safety precautions are given in Chapter 51. 

Editorial Conventions 

(26) For sake of simplicity, abbreviations CI and 1 instead of 
Cl 2 and l 2 are used for chlorine and iodine. Similar abbreviations 
have been used in other cases (0, N, H). The same abbreviation 
may also be used for the ion where no ambiguity will result. 

(27) Reagents and app. referenced with only a letter, e.g., (c), 
will be found in the reagent or apparatus section of that method. 

(28) To conserve space, most of the articles and some prep- 
ositions have been eliminated. 

(29) Names and addresses of manufacturers and suppliers, 
and trade names of frequently mentioned materials, are fur- 
nished below solely as a matter of identification and conven- 
ience, without implication of approval, endorsement, or certifi- 
cation. The same products available from other suppliers or 
other brands from other sources may serve equally well if 
proper tests indicate their use is satisfactory. These firms when 
mentioned in a method are given by name only (without 
addresses). 

Manufacturers and Suppliers 

Ace Glass, lnc. f PO Box 688, 1430 N West Blvd, Vineland, NJ 

08360 
Aldrich Chemical Co., Inc., 940 W St. Paul Ave, Milwaukee, Wl 

53233 
Allied Chemical Corp., Specialty Chemicals Div., PO Box 1087R, 

Morristown, NJ 07960 
Aluminum Company of America, 1501 Alcoa Bldg, Pittsburgh, 

PA 15219 
American Cyanamid Co., Agricultural Div., PO Box 400, 

Princeton, NJ 08540 
American Instrument Co., Div. of Travenol Laboratories, Inc., 

8030 Georgia Ave, Silver Spring, MD 20910 
{ASBQ American Society of Brewing Chemists, 3340 Pilot Knob 

Rd, St. Paul, MN 55121 
{ATCQ American Type Culture Collection, 12301 Parklawn Dr, 

Rockville, MD 20852 
Analabs inc., 80 Republic Dr, North Haven, CT 06473 
Applied Science Laboratories, inc. (Applied Science Division, 

Milton Roy Co.), PO Box 440, State College, PA 16801 
Baird-Atomic, Inc., 125 Middlesex Tnpk, Bedford, MA 01730 
J. T. Baker Chemical Co., 222 Red School Ln, Phillipsburg, NJ 

08865 
Barber-Colman Co., see Searle Analytic, Inc. 
Bausch & Lomb, Inc., Analytical Systems Div., 820 Linden Ave, 

Rochester, NY 14625 
BBL, Div. of Bioquest, PO Box 243, Cockeysville, MD 21030 
Beckman Instruments, Inc., 2500 Harbor Blvd, Fullerton, CA 

92634 
Becton, Dickinson, & Co., Rutherford, NJ 07070 
Bio-Rad Laboratories, 32nd and Griffin Ave, Richmond, CA 94804 
Brinkmann Instruments, Inc., Cantiague Rd, Westbury, NY 1 1590 
Bur dick & Jackson Laboratories, Inc., 1953 S Harvey St, Muske- 
gon, Ml 49442 
Burrell Corp., 2223 Fifth Ave, Pittsburgh, PA 15219 
Calbiochem, 10933 N Torrey Pines Rd, La Jolla, CA 92037 
Carborundum Co., PO Box 423, Niagara Falls, NY 14302 
Cenco Inc., 2600 S Kostner Ave, Chicago, IL 60623 
Coleman Instruments Division, Perkin-Elmer Corp., 2000 York 

Rd, Oak Brook, IL 60521 
Corning Glass Works, Laboratory Products Dept., Corning, NY 

14830 



Curtin Matheson Scientific, Inc., PO Box 1546, Houston, TX 

77001 
Difco Laboratories, PO Box 1058A, Detroit, Ml 48232 
Dohrmann Div. of Envirotech Corp., 3240 Scott Blvd, Santa 

Clara, CA 95050 
Dow Chemical Co., Ag-Organics Dept, PO Box 1706, Midland, 

Ml 48640 
Dow Corning Corp., Midland, Ml 48640 
E. I. du Pont de Nemours & Co., Wilmington, DE 19898 
Eastman Kodak Co., Eastman Organic Chemicals, 343 State St, 

Rochester, NY 14650 
Eaton-Dikeman Co., Mt. Holly Springs, PA 17065 
Eianco Products Co., Div. of Eli Lilly Co., Elanco Analytical 

Laboratory, Dept. MC757, Indianapolis, IN 46206 
Fisher & Porter Co., Lab Crest Scientific Div., County Line Rd, 

Warminster, PA 18974 
Fisher Scientific Co., 711 Forbes Ave, Pittsburgh, PA 15219 
Floridin Co., Berkeley Springs, WV 25411 
Foss America Inc., PO Box 504, Route 82, Fishkill, NY 12524 
GAF Corp., 140 W 51st St, New York, NY 10020 
G.B. Fermentation industries, Inc., 1 N Broadway, Des Plaines, 

IL 60016 
Geigy Chemical Corp., Saw Mill River Rd, Ardsley, NY 10502 
Hamilton Co., PO Box 17500, Reno NV 89510 
Hess <Sr Clark Laboratories, Div. of R hod i a, Inc., 7th and Orange 

Sts, Ashland, OH 44805 
Hewlett-Packard Co., 1501 Page Mill Rd, Palo Alto, CA 94304 
Hoffman-La Roche, Inc., Nutley, NJ 07110 
I CI -Am erica, inc., Chemical Research Dept., Wilmington, DE 

19899 
ICN—K&K Laboratories, Inc., 121 Express St, Plainview, NY 

11803 
ICN Pharmaceuticals, Inc., Life Sciences Group, 26201 Miles Rd, 

Cleveland, OH 44128 
Johns-Manviile Products Corp., Greenwood Plaza, Denver, CO 

80217 
Kimble Products, Owens-Illinois, PO Box 1035, Toledo, OH 43666 
Kontes Glass Co., Spruce St, Vineland, NJ 08360 
Labconco Corp., 8811 Prospect Ave, Kansas City, MO 64132 
Eli Lilly & Co., 740 S Alabama St, Indianapolis, IN 46206 
Mallinckrodt Chemicals Works, Science Products Div., 2nd & 

Mallinckrodt Sts, St. Louis, MO 63147 
MC/B Manufacturing Chemists, 2909 Highland Ave, Norwood, 

OH 45212 
Matheson Scientific, Inc., see Curtin Matheson Scientific, inc. 
Merck & Co., Inc., 126 E Lincoln Ave, Rahway, NJ 07065 
Miles Laboratories, Inc., Elkhart, IN 46514 
Monsanto Chemical Co., 800 N Lindberg Blvd, St. Louis, MO 

63166 
{NBS) National Bureau of Standards, Washington, DC 20234 
(NF) National Formulary, see USP 
New York Laboratory Supply Co., 510 Hempstead Tnpk, West 

Hempstead, NY 11552 
Orion Research Inc., 380 Putnam Ave, Cambridge, MA 02139 
Perkin-Elmer Corp, 702-G Main Ave, Norwalk, CT 06856 
Phillips Chemical Co., Division of Phillips Petroleum Co., Spe- 
cialty Chemicals, Drawer '0', Borger, TX 79007. 
Pierce Chemical Co., PO Box 117, Rockford, IL 61105 
H. Reeve Angel & Co., Inc., 9 Bridewell PI, Clifton, NJ 07014 
Rohm & Haas Co., Independence Mall West, Philadelphia, PA 

19105 
Saisbury Laboratories, Charles City, I A 50616 
Sargent-Welch Scientific Co., 7300 N Linder Ave, Skokie, IL 

60076 
(S&S) Schleicher & Schuell, Inc., 543 Washington St, Keene, NH 

03431 



Schoeffef Instrument Corp., 24 Booker St, Westwood, NJ 07675 
SGA Scientific, Inc., 735 Broad St Bloomfield, NJ 07003 
Scientific Products, Div. of American Hospital Supply Corp., 

1430 Waukegan Rd, McGaw Park, IL 60085 
Searie Analytic, Inc., 2000 Nuclear Dr, Des Plaines, IL 60018 
Shell Oil Co., PO Box 2463, Houston, TX 77001 
Sigma Chemical Co., PO Box 14508, St. Louis, MO 63178 
G. Frederick Smith Chemical Co., PO Box 23344, Columbus, OH 

43223 
Sterwin Chemicals, Inc., 90 Park Ave, New York, NY 10016 
Supeico, Bellefonte, PA 16823 
Technicon Instruments Corp., 51 1 Benedict Ave, Tarrytown, NY 

10591 
Arthur H. Thomas Co., Vine St at 3rd, PO Box 779, Philadelphia, 

PA 19105 
Ultra-Violet Products, Inc., 5100 Walnut Grove Ave, San Gabriel, 

CA 91778 
Union Carbide Corp., Chemicals and Plastics, 270 Park Ave, New 

York, NY 10017 
Union Carbide Corp., Agricultural Products and Services, PO 

Box 1906, Salinas, CA 93901 
Uniroyal Chemical, Elm St, Naugatuck, CT 06770 
The Upjohn Co., Kalamazoo, Ml 49001 
[USDA) U.S. Department of Agriculture, Office of Information, 

Washington, DC 20250 
(USP) United States Pharmacopeial Convention, Inc., 12601 

Twinbrook Pkwy, Rockville, MD 20852 
Marian Aerograph, 2700 Mitchell Dr, Walnut Creek, CA 94598 
Varian instrument Div., 61 1 Hansen Way, Palo Alto. CA 94303 
Vefsicoi Chemical Corp., 341 E Ohio St, Chicago, IL 60611 
VWR Scientific, PO Box 3200, San Francisco, CA 94119 
Wallerstein Co., see G. B. Fermentation Industries, Inc. 
Waters Associates, Inc., Maple St, Milford, MA 01757 
Winthrop Laboratories, Special Chemicals Dept., 90 Park Ave, 

New York, NY 10016 



Trade Names 

Amberlite. Ion exchange resins. Rohm and Haas Co. 

Anakrom, Gas chromatography supports. Analabs, Inc. 

Celite. Diatomaceous products. Johns-Manville Products Corp. 

Chromosorb. Chromatographic supports and packings. Johns- 
Manville Products Corp. 

Dowex. Ion exchange resins, Dow Chemical Co. 

Florisil. Chromatographic adsorbents. Flo rid in Co. 

Gas-Chrom. Gas chromatography solid supports. Applied Sci- 
ence Laboratories, Inc. 

Hyfio Super- Cei. Diatomaceous products. Johns-Manville Prod- 
ucts Corp. 

Skellysolve. Hydrocarbon solvents. Getty Refining and Market- 
ing Co., PO Box 1650, Tulsa, OK 74102 

Teflon. Chemically resistant polytetrafluoroethylene. E. I. du 
Pont de Nemours & Co. 

Tygon. Halogenated vinyl plastic. Norton Co., Plastics & Syn- 
thetics Div., 12 E Ave, Tallmadge, OH 44278 

(30) The folllowing abbreviations, many of which conform 
with those of Chemical Abstracts, are used. In general, principle 
governing use of periods after abbreviations is that period is 
used where final letter of abbreviation is not the same as final 
letter of word it represents. Periods are not used with units, 
except inch{es) and gallon(s). 



Abbreviation 


Word 


a 


absorptivity(ies) 


A 


absorbance(s) thruout (not restricted to for- 




mulas); not absorption. A' is used for std; 




A for blank; 3 digit subscript numerals 




usually denote wavelengths in nm 


AA 


atomic absorption 


Ac 


CH 3 CO- (acetyl, not acetate) 


ACS 


American Chemical Society 


addn 


addition 


addnl 


additional 


ale. 


alcoholic (not alcohol) 


alk. 


alkaline (not alkali) 


alky 


alkalinity 


amp 


ampere(s) 


amt 


amount 


anal. 


analytical(ly) 


anhyd. 


anhydrous 


AOCS 


American Oil Chemists' Society 


app. 


apparatus 


approx. 


approximate(ly) 


aq. 


aqueous 


ASTM 


American Society for Testing and Materials 


atm. 


atmosphere, atmospheric 


a v. 


average (except as verb) 


Be. 


degree Baume 


bp 


boiling point 


Bu 


butyl 


C 


degrees Celsius (Centigrade) 


ca 


about, approximately 


calc. 


calculate 


calcd 


calculated 


calcg 


calculating 


calcn 


calculation 


Cat. No. 


Catalog Number 


centrf. 


centrifuge 


centrfd 


centrifuged 


centrf g 


centrifuging 


Chap. 


Chapter 


chem. 


chemical(ly) 


chromatgc 


chromatographic 


chromatgd 


chromatographed 


chromatgy 


chromatography 


Ci 


curie(s) 


CI 


Color Index 


CIPAC 


Collaborative International Pesticides Ana- 




lytical Council 


cm 


centimeter(s) 


compd 


compound 


com. 


commercial(ly) 


cone. 


concentrate (as verb or noun) 


coned 


concentrated 


concg 


concentrating 


concn 


concentration 


const 


constant 


contg 


containing 


cP 


centi poise 


cpm 


counts per minute 


cry st. 


crystalline (not crystallize) 


crystd 


crystallized 


crystg 


crystallizing 


crystn 


crystallization 


cu in. 


cubic inch(es) 


dc 


direct current 


det. 


determine 


detd 


determined 



Abbreviation 


Word 






Abbreviation 


detg 


determining 






m 


detn 


determination 






M 


diam. 


diameter 






ma 


diat. earth 


diatomaceous earth 






mag. 


dtl. 


dilute 






max. 


dild 


diluted 






mech. 


dilg 


diluting 






Me 


diln 


dilution 






MeOH 


distd 


distilled 






mg 


distg 


distilling 






min 


distn 


distillation 






min. 


DMF 


/V,/V-dimethylformamide 






mixt. 


DMSO 


dimethyl sulfoxide 






ml_ 


EDTA 


ethylenedinitrilotetraacetic 
acetate) 


acid (or 


-tetra- 


mm 
mp 


e.g. 


for example 






m/A 


elec. 


electric(al) 








equiv. 


equivalent 






mv 


est. 


estimate 






MW 


estd 


estimated 






N 


estg 


estimating 








estn 


estimation 






N 


Et 


ethyl 






n 


EtOH 


ethanol (the chemical entity C 2 H 5 OH) 




NBS 


evap. 


evaporate 






NCA 


evapd 


evaporated 








evapg 


evaporating 






neg. 


evapn 


evaporation 






neut. 


ext 


extract 






neutze 


extd 


extracted 






neutzd 


extg 


extracting 






neutzg 


extn 


extraction 






neutzn 


F 


degrees Fahrenheit PC = (5/9) x (°F - 


-32)) 


NF 


FAO 


Food and Agriculture Organization 




ng 


Fig. 


Figure (illustration) 






nm 


fl oz 


fluid ounce(s) (29.57 ml_) 






No. 


fp 


freezing point 






-OAc^ 


ft 


foot (30.48 cm) 






-OCN 


9 


gram(s) 






od 


9 


gravity (in centrfg) 






org. 


gal. 


gallon(s) (3.785 L) 






oxidn 


GLC 


gas-liquid chromatography 






oz 


g-s 


glass-stoppered 






P 


HCHO 


formaldehyde 






Pa 


HOAc 


acetic acid (not HAc) 








HPLC 


high pressure (or performa 


nee) liquid chro- 


par. 




matography 






pet ether 


hr 


hour(s) 






phthln 


ht 


height 






pos. 


id 


inner diameter (or dimension) 




powd 


in. 


inch(es) (2.54 cm) 






ppb 


inorg. 


inorganic 






ppm 


insol. 


insoluble 






ppt 


iR 


infrared 






pptd 


ISO 


International Organization 


for Standardiza- 


pptg 




tion 






pptn 


JAOAC 


Journal of the Association of Official Analyt- 


Pr 




ical Chemists (after 1965) 






prep. 




Journal of the Association 


of Official Agri- 


prepd 




cultural Chemists (before 1966) 




prepg 


kg 


kilogram(s) 






prepn 


L 


liter(s) 






psi 


lb 


pound(s) (453.6 g) 






psig 


liq- 


liquid 








m 


meter(s); milli — as prefix 






pt 



Word 
molal 

molar (as applied to concn), not molal 
milliampere (cf amp) 
magnetic(ally) 
maximum 
mechanical(ly) 
methyl 

methyl alcohol 
milligram(s) 
minute(s) 
minimum 
mixture 
milliliter(s) 
millimeter(s) 
melting point 
millimicron (10~ 6 mm); use nanometer (nm) 

(10- 9 m) 
millivolt 

molecular weight 
normal (as applied to concn); in equations, 

normality of titrating reagent 
Newton (10 5 dynes) 
refractive index 
National Bureau of Standards 
National Canners Association (now National 

Food Processors Association) 
negative 
neutral 
neutralize 
neutralized 
neutralizing 
neutralization 
National Formulary 
nanogram (10~ 9 g) 
nanometer (10~ 9 m); formerly m^t 
number 
acetate (cf Ac) 
cyanate 

outer diameter (or dimension) 
organic 
oxidation 
ounce(s) (28.35 g) 
pico (10~ 12 ) as prefix 
Pascal (1 Newton/m 2 ; 9.87 x 10~ 6 atm.; 7.5 

x 10~ 3 mm Hg (torr); 1.45 x 10" 4 psi) 
paragraph(s) 
petroleum ether 
phenolphthalein 
positive 

powdered (as adjective) 
parts per billion (1/10 9 ) 
parts per million (1/10 6 ) 
precipitate 
precipitated 
precipitating 
precipitation 
propyl 
prepare 
prepared 
preparing 
preparation 

pounds per square inch (absolute) 
pounds per square inch gage (atmospheric 

pressure = 0) 
pint(s) (473 ml_) 



Abbreviation Word 

QAC quaternary ammonium compound 

qt quart(s) (946 mL) 

qual, qualitative(ly) 

quant. quantitative(ly) 

® Trademark name — (Registered) 

8^ distance spot moved/distance solv. moved 
(TLC) 

r-b round-bottom (flask) 

ref. reference 

resp. respectively 

rpm revolutions per minute 

sat. saturate 

satd saturated 

satg saturating 

satn saturation 

-SCN thiocyanate 

SDF special denatured formula (applied to alco- 
hol) 

sec second(s) 

sep. separate(ty) 

sepd separated 

sepg separating 

sepn separation 

sol. soluble 

soln solution 

solv. solvent 

sp gr specific gravity (apparent density) 

spectrophtr spectrophotometer 

spectrophtric spectrophotometric(ally) 

sq square 

SRM Standard Reference Material of National Bu- 
reau of Standards 

std standard 

std dev. standard deviation 

stdzd standardized 

stdze standardize 

stdzg standardizing 

stdzn standardization 

T transmittance 

tech. technical 

temp. temperature 

titr. titrate 

titrd titrated 

titrg titrating 

titrn itration 

TLC thin layer chromatography 

USDA United States Department of Agriculture 



Abbreviation 


Word 


USP 


United States Pharmacopeia 


UV 


ultraviolet 


v 


volt(s) 


v/v 


both components measured by vol. 


vac. 


vacuum 


vol. 


volume; also volumetric when used with 




flask 


w/w 


both components measured by wt 


WHO 


World Health Organization 


wt 


weight 


M 


micron (0.001 mm); use micrometer (//m) 




(10- 6 m) 


M9 


microgram(s) (10~ 6 g) 


/.L 


microliter(s) (10~ 6 L) 


fxrr\ 


micrometer(s) (10~ 6 m); formerly /* 


A 


difference {e.g., AA = {A - A')) 


' 


foot (feet) (V = 30.48 cm) 


n 


inch(es) (1" = 2.54 cm) 


i 


per 


% 


per cent (parts per 100); percentage 


> 


more than; greater than; above; exceeds 




(use with numbers only) 


< 


less than; under; below (use with numbers 




only) 


^ 


not more than; not greater than; equal to or 




less than 


5* 


not less than; equal to or greater than; equal 




to or more than; at least 


f 


standard taper 


f 


standard spherical joint 



(31) * This symbol indicates a method which is in or is being 
considered for "surplus" status. Such methods are satisfactory 
methods, having been subjected to collaborative studies and 
review. They are thought not to be in current use for various 
reasons: The purpose for which they were developed no longer 
exists; the product for which they were developed no longer is 
marketed; they have been replaced by other methods; etc. 
These methods retain their official status but are carried in this 
or next edition only by ref. Any laboratory who uses these 
methods and wishes the text retained or reprinted in next edition 
must so notify the AOAC. 

(32) Nos. appearing in titles of methods in bold face refer to 
the Selected References at the end of the Chap. These refs often 
contain the performance data supporting the adoption of the 
method. 



xx 



1. Agricultural Liming Materials 



1.001 Sampling (?) — Procedure 

{Caution: See 51.036.) 

Take sample representative of lot or shipment. Avoid dispro- 
portionate amt of surface or any modified or damaged zone. 

(a) Burnt or lump lime, in bulk. — Collect composite sample of 
5*10 shovelfuls/car, with proportionate amts from smaller lots, 
taking each shovelful from different part of lot or shipment. 
Immediately crush to pass 5 cm (2") diam. circular opening, mix 
thoroly and rapidly, reduce composite to ca 2 kg {5 lb) sample 
by riffling or quartering, and place in labeled, dry, air-tight 
container. 

(b) Hyd rated lime and ground burnt lime, in bags. — Select 10 
bags from different parts of each lot or shipment of *s20 tons 
and 1 addnl bag for each addnl 5 tons. Use sampling tube to 
withdraw top to bottom core from each bag selected. Combine 
cores, mix thoroly and rapidly, reduce composite to ca 1 kg (2 
lb) by riffling or quartering, and place in dry, air-tight container. 

(c) Ground limestone and ground marl, in bags.— Proceed as 
in (b). 

(d) Ground limestone, ground burnt lime, ground marl, and 
slag, in bulk. — Use slotted sampling tube to withdraw samples 
to full sampler depth from 10 points in lot or shipment. Proceed 
as in (b), beginning "Combine cores, . . ." 

1.002 Mechanical Analysis (2) — Procedure 

(Caution: See 51.036.) 

If entire sample is not to be dried, obtain lesser portions by 
riffling or quartering. Dry at 110° to const wt and cool to room 
temp. 

Obtain 90-150 g dry sample by riffling or quartering. Break 
any agglomerates formed during drying by rolling dry sample 
with hard rubber roller on hard rubber mat, wet sieving as in 
2.011(a), or by equally effective means that does not result in 
crushing the limestone. (If wet sieving is used to break agglom- 
erates, do wet sieving on sieve having smallest opening to be 
used in final testing. After drying, transfer to sieves to be used 
in final testing. If only 1 sieve is to be used, do not transfer.) 
Quant, transfer weighed sample to 8" diam. std sieve or set of 
sieves (e.g., Nos. 10, 20, 40, 60, 80, and 100 or other appropriate 
combination). 

Sieve by lateral and vertical motion accompanied by jarring 
action. Continue 5=5 min or until addnl 3 min of sieving time 
fails to change results of any sieve fraction by 0.5% of total 
sample wt. Do not overload any sieve when assaying closely 
sized materials. 

Det. wt of each sieve fraction and report as % of total sample 
wt. 

1.003 Preparation of Sample (7) — Procedure 

Reduce dried sample, 1.002, to amt sufficient for analysis and 
grind ^225 g (0.5 lb) reduced sample in mortar, ball mill, or 
other mech. app. to pass No. 60 sieve. Mix thoroly, and store 
in air-tight container. 

Neutralizing Value — Official Final Action 

(Uncorrected for sulfide content) 



1.004 



Reagents 



(b) Hydrochloric acid std soln. — 0.5/V. Stdze against (a), using 
phthln. 

1 .005 Indicator Titration Method 

Place 0.5 g burnt or hydrated lime (1 g ground limestone or 
ground marl), prepd as in 1.003, in 250 mL erlenmeyer; add 50 
mL HCI std soln and boil gently 5 min. Cool, andtitr. excess acid 
with NaOH std soln, using phthln. For burnt and hydrated lime, 
report as % CaO; for limestone and marl, report as % CaC0 3 
equivalence. 

% CaC0 3 equivalence of sample = 2.5 x (mL HCI - mL 

NaOH/2). 

% CaO equivalence - 2.8 x (mL HCI - mL NaOH/2). 

1 .006 Potentiometric Titration Method {3) 

(Applicable to liming materials contg large amt of Fe +2 or 
coloring matter, but not to silicate materials) 

Proceed as in 1.005 thru ''Cool, . . ." Transfer to 250 mL beaker 
and insert glass and calomel electrodes of pH meter, buret contg 
0,25/V NaOH, and mech. stirrer. Stir at moderate speed to avoid 
splash. Deliver NaOH rapidly to pH 5, then dropwise until soln 
attains pH 7 and remains const 1 min while stirring. (If end point 
is passed, add, from 1 mL Mohr pipet, just enough 0.5/V HCI to 
bring pH to <7, and back-titr. slowly to pH 7.) Add mL of excess 
acid, if used, to initial 50 mL in calcg. Report as % CaC0 3 or CaO 
equivalence as in 1.005. 

1 .007 Approximate Proportions of Calcium 
and Magnesium in Magnesic Limestone 

Slightly acidify titrd soln, 1.005 or 1.006, transfer to 250 mL 
vol. flask, and dil. to vol. Det. Ca in 50 mL aliquot as in 7.096, 
beginning ". . . dil. to ca 100 mL . . ." Subtract its CaC0 3 
equivalence from total CaC0 3 equivalence, 1.005 or 1.006, and 
assign difference as CaC0 3 equivalence of the Mg content of the 
limestone. 



1.008 



Caustic Value {4)—- Official Final Action 

Apparatus {Figure 1:01) 



Use 500 mL Pyrex erlenmeyer, A, and fritted glass filter 
(Corning Glass Works No. 39535, 30F),f. Connect filter to siphon 
tube B with thick-wall rubber tubing. Use receiving flasks M and 
N calibrated to deliver 50 and 100 mL, res p. S is suction flask. 



1.009 



Determination 



(a) Sodium hydroxide std soln. — 0.25/V. Prep, and stdze as in 
50.032-50.036. 



Transfer portion of sample, 1.003, to weighing bottle and det. 
wt bottle and contents in atm. of min. moisture and C0 2 content. 
With polished, narrow-point spatula calibrated to hold ca 1.5 g, 
withdraw sample to be used and det. exact wt by difference. 
Insert sample directly into dry flask, A, fitted with tight rubber 
stopper. 

Prep, sucrose soln immediately before use by placing 25 g 
granulated sucrose in measuring flask calibrated to deliver 500 
mL. Dissolve sucrose with cold CO^free H 2 and dil. to vol. 
Holding both erlenmeyer contg sample and flask contg sucrose 
soln in slightly inclined position, insert neck of sucrose soln flask 
short distance into erlenmeyer, and carefully transfer sucrose 
soln with synchronized rotary motion of both flasks to prevent 
granulation of lime. Stopper erlenmeyer securely, agitate, and 



1 . Agricultural Liming Materials 



AOAC Methods (1980) 



add, if desired, some clean dry beads. Completely dissolve 
uncoated caustic lime by six 1 min agitations at 2 or 3 min 
intervals. Invert flask to trap any solid particles between stopper 
and neck and crush by carefully twisting stopper. Let stand 15 
min and filter as follows: 

Connect filter cone F with siphon B and close stopcock D. 
Connect receiving flasks, apply suction, and quickly connect 
erlenmeyer>4 contg lime soln with stopper E. Open stopcock C 
and filter 25-50 mL soln. Close C and open D to release suction. 
Remove M and replace with similar dry flask. Close D, open C, 
and continue filtration until both M and N are filled at least to 
marks. To disconnect system, close stopcock C, and gently press 
down outlet of flask M and then outlet of flask N, to remove any 
excess liq. above marks. Let intermediate connection empty, 
open stopcock D, and remove M and A/. Titr. first 50 mL, or pilot 
aliquot, of filtered soln with 0.5/V HCI, using phthln. To covered 
200 mL beaker add twice vol. 0.5A/ acid required for this titrn, 
add second (100 mL) aliquot of filtered soln to this acid and 
phthln, and complete titrn. 

Calc. caustic value of sample: X = IV /W, 
where X - % active CaO; V = mL 0.5/V acid used/100 mL lime 
soln; W = g sample. 



Carbon Dioxide (5) — Official Final Action 



1.010 



Apparatus and Reagents 



Knorr alkalimeter with C0 2 absorption train. — Fill guard tube 
of alkalimeter with Ascarite. Connect upper end of condenser 
to absorption train consisting of 5 or 6 U-shape, g-s drying tubes 
(or equiv.) joined in series. Fill first tube with H 2 S0 4 and second 
with Ag 2 S0 4 -H 2 S04 soln (10 g Ag 2 S0 4 in 100 m.L H 2 S0 4 )to remove 
acidic gases other than C0 2 . Fill third tube with Mg(CI0 4 ) 2 to 
absorb H 2 0. Fill inlet 2 / 3 of fourth and succeeding tubes with 
Ascarite to absorb C0 2 , and outlet V 3 of each tube with Mg(CI0 4 ) 2 . 
Connect last tube in train with aspirating bottle or suction 
source. 



Condition app. daily before use, and also when freshly filled 
tube is placed in train, by aspirating air at rate of 2-3 bubbles/sec 
thru dry alkalimeter assembly and absorption train until C0 2 
absorption tubes attain const wt (usually 20-30 min). Tare 
against similarly packed tubes. Use std procedure for wiping 
tubes with dry, lint-free cloth before each weighing. 



1.011 



Determination 



Transfer 3 g burnt or hydrated lime or 0.5-1.0 g limestone or 
marl, prepd as in 1.003, to dry alkalimeter flask. Momentarily 
open stopcocks of first 2 C0 2 absorption tubes to air to equalize 
pressure, weigh tubes sep., and place in position in train. With 
assembled alkalimeter connected to absorption train, adjust rate 
of aspiration of air thru system to ca 2 bubbles /sec. Close funnel 
stopcock, remove alkalimeter guard tube, fill funnel with 50 mL 
HCI (1+4), and replace guard tube. Open funnel stopcock and 
let acid run slowly into flask, taking care that evolution of gas 
is so gradual as not to materially increase flow thru tubes. After 
all acid is added, agitate alkalimeter assembly to ensure com- 
plete dispersion of sample in acid soln. Continue aspiration, 
gradually heat contents of flask to bp, and boil 2-3 min after 
H 2 begins to condense. Discontinue heating, and continue 
aspiration 15-20 min or until app. cools. Remove, equalize 
internal and external pressure, and reweigh absorption tubes. 

Increase in wt == wt C0 2 . (Material increase in wt of second 
tube usually indicates exhaustion of first tube, but may result 
from too rapid evolution of C0 2 in relation to aspiration rate.) 
Report % CaC0 3 . 

CALCIUM SILICATE SLAGS 

1.012 Neutralizing Value {6) — Official Final Action 

(Uncorrected for sulfide content) 

(a) Blast furnace slag. — Transfer 0.5 g sample, ground to pass 
No. 80 sieve, to 250 mL erlenmeyer. Wash down with small 




FIG. 1:01— Apparatus for automatic filtration and measurement of lime solutions 



AOAC Methods (1980) 



Elemental Analysis 



portions H 2 and add 35 mL 0.5/V HCI while swirling. Heat to 
gentle boil over burner, agitating suspension continuously until 
bulk of sample dissolves. Boil 5 min and cool to room temp.; 
then dil. with C0 2 -free H 2 to ca 150 mL and add 1 mL30% H 2 2 
and 5 drops bromocresol green, 2.144(c). Back-titr. with 0.5/V 
NaOH, adding first 15 mL rapidly and titrg dropwise thereafter, 
vigorously agitating contents of stoppered flask after each addn, 
until indicator tint matches or slightly exceeds that of pH 5.2 
phthalate buffer soln, 50.010, of like vol. and indicator concn, 
after 2-3 sec agitation. 

(b) Rock phosphate reduction furnace slag. — Transfer 0.5 g 
sample to 250 mL beaker. Wash down with small portions H 2 
and add, stirring continuously, 50 mL HOAc (1+4). Heat to bp 
and boil 5 min, stirring frequently. Evap. to dryness on steam 
bath. Add 20 mL of the HOAc, dil. to 150 mL, and heat to bp; 
add NH 4 OH (1+1) to distinct yellow of Me red. Digest ca 10 min 
on hot plate. Filter by gravity thru 9 cm paper, catching filtrate 
in 100 x 50 mm lipped Pyrex crystg dish; wash beaker 3 times 
and paper 5 addnl times with neut 0.5N NH A OAc. Evap. filtrate 
on hot plate. Adjust heat so bubbles breaking thru viscous 
surface film are released gently to avoid spattering. (To expedite 
dehydration, repeat treatments with 25 mL hot H 2 and evapn 
2 or 3 times.) Continue heating residue on hot plate until no 
HOAc odor remains. Heat addnl 10 min at full heat of hot plate; 
then ignite 10 min at 550°. Cool, wet residue with 15 mL H 2 0, 
place watch glass over dish, and add 25 mL 0.5/V HCI thru lip 
of dish. Heat 5 min over burner at gentle simmer. Rinse watch 
glass, filter suspended matter on 9 cm paper, catching filtrate 
in 250 mL erlenmeyer, and wash dish and filter 3 times with hot 
H 2 0. Titr. excess acid with 0.5/V NaOH to distinct yellow of Me 
red. 

Net acid used x 5 = neutzg value of slag in terms of % CaC0 3 
equivalence. 

Sulfide Sulfur (7)— Official Final Action 
(Note: CdS0 4 is toxic: see also 51.084.) 

1.013 Reagents 

(a) Zinc dust. — Low in Pb. 

(b) Absorbent soln. —Dissolve 20 g CdS0 4 .2 2 / 3 H 2 in H 2 and 
dil. to 1 L. Adjust to pH 5.6 potentiometrically orcolorimetrically. 
If colorimetrically, match sep. 50 mL aliquot to buffer of same 
pH, 50.010. 

(c) Sodium hydroxide stdsoln.—QAN. Prep, and stdze as in 
50.032-50.036. 

(d) Stdacid.—QAN HCI. Stdze against std alkali, (c), using Me 
red. 

(e) Methyl red indicator. — Dissolve 0.2 g Me red in 100 mL 
alcohol. 

1.014 Apparatus 

Fit 250 mL erlenmeyer with 2-hole No. 5.5 stopper. Insert thru 
stopper 60 mL separator with stem drawn out to 2 mm and bent 
upward at tip, adjusting separator so stem is 6 mm from bottom 
of flask. Also insert thru stopper 6 mm glass outlet tube. Connect 
with amber rubber tubing to inlet of 25 x 150 mm tube half 
filled with H 2 and heated to near bp before and during detn. 
Connect in series 2 addnl tubes of same size, each contg 25 mL 
absorbent soln and held in 600 mL beaker filled with cold H 2 0. 

1 .01 5 Determination 

Fill absorbent tubes with absorbent soln and heat H 2 tube 
to gentle boiling. Weigh 1 g slag, ground to pass No. 80 sieve, 
into evolution flask, add 1 g Zn dust, and wash down sides with 
5-10 mL H 2 0; mix with flat-end rod and connect flask to app. 



Add 50 mL HCI (1 +4) to separator and let acid flow into reaction 
flask while swirling contents. If necessary, apply pressure to 
transfer acid and close stopcock while a little of the acid is still 
above it. Heat to bp; then regulate to maintain active but not 
too vigorous boiling for 10 min. Swirl flask frequently after 
adding acid and for first 5 min of boiling. To disconnect, hold 
inlet in first absorbent tube firmly with one hand and quickly 
pull off rubber tubing with other hand without pinching. 

Filter CdS suspension by gravity thru 9 cm paper into 250 mL 
erlenmeyer and wash with H 2 to vol. of 100 mL. Add 4 drops 
Me red indicator and agitate vigorously while titrg slowly with 
0.1/V NaOH to exact tint of ref. soln (50 mL absorbent soln dild 
to 100 mL, with identical indicator concn, in 250 mL erlenmeyer). 
If end point is passed so that Cd(OH) 2 ppts, add 1-2 mL 0.1/V 
HCI, let stand until ppt disappears, and complete titrn dropwise, 
agitating vigorously. 

% CaC0 3 equivalence of sulfide 5 in sample = net mL 0.1/V 
NaOH/2 

g Sulfide S/detn - mL 0.1/V NaOH x 0.0016 

% Sulfide S - g sulfide S x 100 

ELEMENTAL ANALYSIS 
Gravimetric Methods 

1.016 Preparation of Sample Solution by Acid 
Digestion (5)— Official Final Action 

[Caution: See 51.019, 51.026, and 51.028.) 
Prep, samples as in 1.003, preferably in agate mortar. Grind 
silicates to pass No. 100 sieve, and dry all samples at 105°. 

Weigh 2 g limestone or 0.5 g silicate. If sample contains org. 
matter, transfer to Pt crucible and place in cold furnace. Raise 
temp, gradually to 1000° and hold 15 min. Transfer sample to 
400 mL beaker and, if ignited, moisten cautiously with H 2 0. Add 
10 mL HNO3 and evap. on hot plate at low heat until mixt. 
becomes pasty. Cool, and add 10 mL H 2 and 20 mL 60% HCI0 4 . 
Boil to heavy fumes of HCI0 4 , cover, and fume slowly until soln 
is colorless or slightly yellow (5-10 min). Do not evap. to 
dryness. Cool to <100° and add 50 mL H 2 0. Filter thru Whatman 
41 H or finer paper into 250 mL vol. flask. Wash thoroly with hot 
H 2 to remove ail traces ofHCI0 4 . Reserve filtrate and washings 
for prepn of Sample Solns X and Y, 1.017(a) and (b). 

1.017 Silica [8) — Official Final Action 

{See also 1.041-1.043.) 
{Caution: See 51.025 and 51.028.) 

Transfer paper with Si0 2 to uncovered Pt crucible and heat 
gently with low flame until paper chars without flame. Partially 
cover crucible and cautiously burn C. Finally cover completely 
and heat with blast lamp or in furnace at 1150-1200°. Cool in 
desiccator and weigh. Repeat to const wt (W). Treat with ca 1 
mL H 2 0, 2 drops H 2 S0 4 (1+1), and 10 mL HF. Cautiously evap. 
to dryness in hood. Heat 2 min at 1050-1 100°, cool in desiccator, 
and weigh (B). 

W - B = g Si0 2 in sample, 
g Si0 2 x 0.4674 - g Si. 

(a) Sample Soln X.-(0.008 g limestone or 0.002 g silicate/mL.) 
Fuse residue from Si detn with 0.5 g Na 2 C0 3 by heating covered 
crucible 10 min over Meker burner. Cool, fill crucible 2 / 3 full with 
H 2 0, and add 2 mL 60% HCI0 4 dropwise, with stirring. Warm if 
necessary to dissolve melt. Add to filtrate and washings reserved 
for prepn of Sample Soln X in 1.016. Dil. to 250 mL with H 2 0. 

(b) Sample Soln /.-(0.00016 g limestone or 0.00004 g 
silicate/mL.) Dil. 10 mL Sample Soln X to 500 mL with H 2 0. 



1. Agricultural LfMiNG Materials 



AOAC Methods (1980) 



1.018 Oxides of Iron, Aluminum, Phosphorus, 
and Titanium (9) — Official Final Action 

(Alternatively, Fe, Al, Mn, P, and Ti may be detd 
colorimetrically as in 1.025-1.040.) 

To 125 mL aliquot Soln X from 1.017(a), add 10 mL HCI and 
few drops Me red indicator; heat to gentle boil and add NH 4 OH 
(1+1) until ppt forms and indicator just changes to distinct 
yellow. Boil ^2 min and filter rapidly. Wash ppt 6-8 times with 
hot 2% NHJVOz soln. Return ppt and filter to original beaker, 
add 10 mL HCI, and macerate filter with policeman. Dil. with 
H 2 0, heat to dissolve ppt, dil. to ca 200 mL, and reppt as above. 
Wash thoroly with the hot NH 4 N0 3 soln until Chfree. Combine 
first and second filtrates and save for Ca and Mg detns. 

Place ppt in Pt crucible and dry. Ignite gently to oxidize C, 
heat to bright red ca 10 min, cool in desiccator, and weigh in 
covered crucible as Fe 2 3 + Al 2 3 + P 2 5 + Ti0 2 . 



1.019 Calcium [9)— Official Final Action 

Cone, combined filtrates and washings from 1.018 to ca 50 
mL; make slightly alk. with NH 4 OH (1 + 1); while still hot, add 
satd (NH 4 ) 2 C 2 4 soln dropwise as long as any ppt forms, and 
then enough excess to convert Mg salts also to oxalate. Heat to 
bp, let stand ^3 hr, decant clear soln thru filter, pour 15-20 mL 
hot H 2 on ppt, and again decant clear soln thru filter. Dissolve 
any ppt remaining on filter by washing with hot HCI (1 +9) into 
original beaker, wash 6 times with hot H 2 0, and then reppt at 
bp by adding NH 4 OH and a little satd (NH 4 ) 2 C 2 4 soln. Let stand 
as before, filter thru same filter, and wash with hot H 2 until Cl- 
free. Reserve filtrates and washings from both pptns for detn of 
Mg, 1.021. 

Complete detn by one of following methods and report as 
% CaO: 

(a) Ignite ppt in crucible, either over S-free blast lamp, or in 
elec. furnace at 950°, to const wt, cool in desiccator, and weigh 
as CaO. 

(b) Incinerate filter over low flame, mix ignited ppt with finely 
pulverized and dried mixt. of equal parts of {NH 4 ) 2 S0 4 and NH 4 Cf, 
and drive off excess sulfate by carefully heating upper portion 
of crucible. Complete ignition, cool in desiccator, and weigh as 
CaS0 4 . 

(c) Perforate apex of cone; wash CaC 2 4 ppt into beaker used 
for pptn; then wash filter with hot H 2 S0 4 (1 +4), and titr. at 85-90° 
with 0.1/V KMn0 4 . 



Magnesium (70>— Official Final Action 

1 .020 Reagent 

Phosphate soln.— Dissolve 100 g (NH 4 ) 2 HP0 4 in hot H 2 0, dil. 
to 1 L, and add 5 mL CHCI 3 . 



1.021 



Determination 



To combined filtrates and washings, 1.019, add 2 mL MVI citric 
acid, 100 mL NH 4 OH, and 50 mL alcohol. Then add 25 mL of the 
phosphate soln, with const stirring, and let stand 12-24 hr. 
Filter, wash twice with NH 4 OH (1+9), and dissolve ppt in HN0 3 
(1 +4), washing soln into original beaker to vol. of 100-150 mL. 
Add 1/10 vol. IMH 4 OH and 2 drops of the phosphate soln. Stir 
vigorously and let stand ^3 hr. Filter thru gooch, wash with 
NH 4 OH (1+9), moisten filter with satd soln of NH 4 N0 3 made 
slightly ammoniacal, ignite, and weigh as Mg 2 P 2 7 . Report as 
% MgO. Correct wt Mg 2 P 2 7 for co-pptd Mn 2 P 2 7 by detg Mn as 
in 33.127. 



EDTA Titration Methods 

Calcium and Magnesium {11} — Official Final Action 

(Not applicable to samples with high 
phosphate content or contg <2% Mg) 

{Caution: See 51.050.) 
1.022 Reagents 

(a) Buffer so/a?.— pH 10. Dissolve 67.5 g NH 4 CI in 200 mL H 2 0, 
add 570 mL NH 4 OH, and dil. to 1 L. 

(b) Potassium hydroxide-potassium cyanide soln. — Dissolve 
280 g KOH and 66 g KCN in 1 L H 2 0. 

(c) Potassium cyanide soin. — 2%. Dissolve 2 g KCN in 100 
mL H 2 0. 

(d) Eriochrome black T indicator soin. — Dissolve 0.2 g indi- 
cator (Eastman Kodak P6361, or equiv.) in 50 mL MeOH contg 
2 g NH 2 OH.HCI. Store *=1 month. 

(e) Magnesium std solns. — 0.25 and 1.00 mg/mL Dissolve 
0.25 and 1.00 g Mg turnings in HCI (1+10) and dil. each to 1 L 
with double distd H 2 0. 

(f) Calcium std sofn.—l mg/mL. Dissolve 2.4973 g CaC0 3 , 
primary std grade, previously dried 2 hr at 285°, in HCI (1 + 10). 
Dil. to 1 L with double distd H 2 0. 

(g) Calcein indicator. — Grind together 1 g indicator, 10 g 
charcoal (Norite A is satisfactory), and 100 g KCI. (Indicator is 
described in Anal. Chem. 28, 882 (1956), and is available from 
G. Frederick Smith Chemical Co. and Eastman Kodak.) 

(h) Dis odium di hydrogen EDTA std solns. — (7) 0.4%. — Dis- 
solve 4 g Na 2 H 2 EDTA in 1 L H 2 0. Stdze against std Ca and Mg 
solns. {2)0.7%.— Prep, as in (7), using 1 g Na 2 H 2 EDTA, and stdze 
against 0.25 mg/mL Mg std soln. 



1.023 



Standardization 



(a) For calcium.— Pipet 10 mL std Ca soln into 300 mL erlen- 
meyer and add 10 mL H 2 0. Add 10 mL KOH-KCN soln and ca 35 
mg calcein indicator. Using mag. stirrer and artificial light, titr. 
with 0.4% EDTA std soln to disappearance of all green. Titr. ^3 
aliquots and use av. to calc. titer Ca soln = 10/mL EDTA soln. 

(b) For magnesium. — Pipet 10 mL 0.25 and 1.00 mg/mL Mg 
std solns into 300 mL erlenmeyers and add 100 mL H 2 0. Add 5 
mL pH 10 buffer, 2 mL 2% KCN soln, and 10 drops eriochrome 
black T indicator. Using mag. stirrer and artificial light, titr. with 
0.1 and 0.4% EDTA std solns, resp., until color changes per- 
manently from wine red to pure blue. Titr. ^3 aliquots and use 
av. to calc. titer Mg soln = 2.5/mL EDTA soln, or 10/mL EDTA 
soln, resp. 



1.024 



Determination 



Dry sample at 110° to const wt and cool to room temp. Grind 
to pass No. 60 or 80 sieve and mix thoroly. Accurately weigh ca 
0.5 g into 250 mL beaker, add 20 mL HCI (1+1), and evap. to 
dryness on hot plate. Dissolve residue in 5 mL HCI (1 + 10), dil. 
to ca 100 mL with H 2 0, and digest over low flame 1 hr. Cool, 
transfer to 200 mL vol. flask, dil. to vol., mix, and let settle or 
filter. 

(a) For calcium. — Pipet 10 mL aliquot into 300 mL erlenmeyer 
and titr. as in 1.023(a), observing end point thru soln and away 
from light. % Ca - (Titer EDTA std soln for Ca) x mL EDTA std 
soln x 2/g sample. 

(b) For magnesium. — (For agricultural limestones contg >4% 
Mg.) For Ca + Mg, pipet 10 mL aliquot into 300 mL erlenmeyer 
and titr. with 0.4% EDTA soln as in 1.023(b). 

o/ Mg = (Titer EDTA std soln for Mg) x [(mL EDTA std soln 
in Ca + Mg titrn) - (mL EDTA std soln in Ca titrn)] x 2/g sample. 

(c) For magnesium.— (For agricu It ura I limestones contg 2-4% 



AOAC Methods (1980) 



Elemental Analysis 



Mg.) Pipet 10 mL aliquot (0.5-1 .0 mg Mg) into 300 ml_ erlenmeyer 
and add exact vol. of 0.4% EDTA soln required for Ca detn. Titr. 
with 0.1% EDTA soln as in 1.023(b). 

% Mg - (Titer EDTA std soln for Mg) x mL EDTA std 0.1% 
soln x 2/g sample. 

Colorimetric Methods ( 72)— Official Final Action 

(Carry reagent blanks thru detn with stds and samples. Treat 
aliquots of blank soln (corresponding to aliquot sizes of sample 
solns taken for analysis) as in Determination for appropriate 
element and correct values for samples accordingly.) 

Det. Al, Fe, Mn, P, and Ti in solns prepd by HCI0 4 digestion, 
1.016-1.017, or NaOH fusion, 1.025. Det. Si only in soln prepd by 
NaOH fusion. 

1.025 Preparation of Sample Solution 
by Sodium Hydroxide Fusion 

Prep, samples as in 1.003, preferably in agate mortar. Grind 
samples to pass No. 100 sieve and dry at 105°. 

(a) Sample Soln X— (0.005 g limestone or 0.002 g silicate/mL.) 
Place 0.5 g limestone or 0.2 g silicate in 75 mL Ni crucible. If 
sample contains org. matter, place uncovered crucible in cold 
furnace, raise temp, gradually to 900°, and hold 15 min. Remove 
crucible from furnace and let cool. Mix 0.3 g KN0 3 with sample 
and add 1.5 g NaOH pellets. Cover crucible with Ni cover and 
heat 5 min at dull redness over gas flame. (Do not fuse in 
furnace.) Remove from flame and swirl melt around sides. Cool, 
add ca 50 mL H 2 0, and warm to disintegrate fused cake. Transfer 
to 150 mL beaker contg 15 mL 5/V HCI0 4 (1(60%)+1). Scrub 
crucible and lid with policeman, and wash any residue into 
beaker. Transfer to 1 00 mL vol. flask and dil. to vol. {Sample Soln 
X). (This soln is acidic and is normally clear and free of insol. 
matter. Occasionally particles of oxidized Ni from crucible 
appear. When this occurs, let particles settle before taking 
aliquots.) 

(b) Sample Soln /. —(0.0001 5 g limestone or 0.00004 g 
silicate/mL.)Dil.15mLlimestoneSa/r7p/eSo/A7Xor10mLsiltcate 
Sample Soln X to 500 mL with H 2 0. 



Aluminum 



1.026 



Reagents 

(a) Aluminum std solns.— {1) Stock soln. — 100 /xg AI/mL. To 
0.1000 g pure Al metal in 30 mL beaker, add 6 mL HCI (1 + 1). 
Cover with watch glass and warm gently until Al completely 
dissolves. Dil. to 1 L with H 2 0. {2) Working soln.— A ng AI/mL. 
Dil. 20 mL stock soln to 500 mL. 

(b) Aluminon soln, — Dissolve sep. in H 2 0: 0.5 g NH 4 aurintri- 
carboxylate in 100 mL; 10 g acacia (gum arabic) in 200 mL; and 
100 g NH 4 0Ac in 400 mL. Filter acacia soln. Add 56 mL HCI to 
NH 4 OAc soln and adjust pH to 4.5 with HCI or NH 4 0H. Combine 
3 solns and dil. to 1 L with H 2 0. 

(c) Antifoam soln. — Disperse 0.03 g silicone defoamer (Dow 
Corning Corp. Antifoam A) in 100 mL H 2 0. 

(d) Thioglycolic acid soln.— DM 1 mL HSCH 2 COOH to 100 mL 
with H 2 0. 

1 027 Preparation of Standard Curve 

Transfer aliquots of std soln contg 0, 4, 20, 40, 60, and 80 /xg 
Al to 100 mL vol. flasks and proceed as in detn. Prep, std curve 
by plotting %T against /xg Al on semilog paper. 

1 .028 Determination 

Use Sample Soln X for limestones contg <0.2% or silicates 



contg <0.8% Al and adjust pH of aliquot to 4.5 with NH 4 0H. For 
materials contg greater concns of Al, use Sample Soln /and omit 
pH adjustment 

Transfer aliquot (=£20 mL contg <80 /xg Al) of Sample Soln X 
or Y to 100 mL vol. flask. Dil. to 20 mL with H 2 0. Add 2 mL 
thioglycolic acid soln, 0.5 mL antifoam soln, and 10 mL aluminon 
soln. Place flask in boiling H 2 20 min (250 mL beaker contg 125 
mL H 2 holds 100 mL vol. flask conveniently). Remove flask 
from H 2 and let cool ca 30 min. Dil. to 100 mL with H 2 0. Use 
/xg Al soln, 1.027, to set 100% 7" at 525 nm. Read %T for 
sample soln and det. fig Al from std. curve. Calc. % Al in sample. 



Iron 



1.029 



Reagents 



(a) Iron std solns. — {/) Stock soln. — 100 /xg Fe/mL. Dissolve 
0.1000 g pure Fe metal in 5 mL IN HCI and dil. to 1 L with H 2 0. 
(2) Working soln.—S fig Fe/mL. Dil. 25 mL stock soln to 500 mL. 

(b) 2,4,6-Tripyridyl-s-triazine {TPTZ) soln.— (Available from 
G. Frederick Smith Chemical Co.) Dissolve 0.500 g TPTZ in few 
drops HCI and dil. to 1 L with H 2 0. 

(c) Hydroxylamine hydrochloride soln. — Dissolve 50 g 
NH 2 OH.HCI in H 2 0. Add 10 mLTPTZ soln and 0.5 g NaCI0 4 .H 2 0, 
and dil. to 500 mL with H 2 0. Transfer to separator, add 25 mL 
nitrobenzene, and shake several min. Let phases sep. and 
discard lower nitrobenzene phase contg Fe. Repeat extn 2 or 3 
times. 

(d) Acetate buffer soln. — Dissolve 164 g anhyd. NaOAc in 
H 2 0. Add 115 mL HOAc, 10 mL NH 2 OH.HCI soln, 0.05 g TPTZ, 
and 1 g NaCI0 4 .H 2 0, and dil. to 1 L with H 2 0. Transfer to 
separator, add 25 mL nitrobenzene, and shake several min. Let 
phases sep. and discard lower nitrobenzene phase. Repeat extn 
3 or 4 times. 

1.030 Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 100 fig Fe as in 
detn. Prep, std curve by plotting %T against fig Fe on semilog 
paper. 



1.031 



Determination 



Use Sample Soln X (^5 mL) for limestones contg <0.05% or 
silicates contg <0.2% Fe and Sample Soln Y for materials contg 
greater concns of Fe. 

Transfer aliquot (<1 00 /xg Fe) of Sample Soln X or Y to 100 mL 
vol. flask. Add 3 mL NH 2 OH.HCI soln and 10 mL TPTZ soln. Add 
NH 4 OH dropwise until Fe derivative remains violet on mixing. 
Add 10 mL buffer soln and dil. to 100 mL. Use /xg Fe soln, 
1.030, to set 100% T at 593 nm. Read %T for sample soln and 
det. fig Fe from std curve. Calc. % Fe in sample. 



Manganese 

1.032 Reagents 

(a) Manganese std soln. — 50 fig Mn/mL Dissolve 0.0500 g 
pure Mn metal in 20 mL 0.5/V H 2 S0 4 and dil. to 1 L with H 2 0, 

(b) Acid mixture.— Add 800 mL HN0 3 and 200 mL H 3 P0 4 to 
H 2 and dil. to 2 L. 

1 .033 Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 50, 100, 300, and 500 fig Mn 
as in detn. Prep, std curve by plotting %T against /xg Mn on 
semilog paper. 



1 . Agricultural Liming Materials 



AOAC Methods (1980) 



1.034 



Determination 



Transfer aliquot (<500 /xg Mn) of Sample Soln X to 150 mL 
beaker. Add 25 mL acid mixt. and 0.3 g Ki0 4 . Bring to bp and 
keep near boiling temp. 10 min after color develops. Let cool, 
transfer to 50 mL vol. flask, dil. to vol., and mix. Use /xg Mn 
soln, 1.033, to set 100% 7" at 525 nm. Read %T for sample soln 
and det. fxg Mn from std curve. Calc. % Mn in sample. 

Phosphorus 

(Do not clean glassware with detergents contg P.) 

1 .035 Reagents 

(a) Phosphorus std so Ins. — (1) Stock soln. — 100 /xg P/mL. 
Dissolve 0.4393 g KH 2 P0 4 in H 2 and dil. to 1 L (2) Working 
soln. — 5 fxg P/mL. Dil. 25 mL stock soln to 500 mL. 

(b) Ammonium molybdate soln. — Dissolve 20 g 
(NH 4 ) 6 Mo 7 24 .4H 2 in 500 mL H 2 0. Add 285 mL H 2 S0 4 , cool, and 
dil. to 1 Lwith H 2 0. 

(c) Hydrazine sulfate soln. — Dissolve 2 g N 2 H 4 .H 2 S0 4 in H 2 
and dil. to 1 L. 

1 .036 Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 75 /*g P as in 
detn. Prep, std curve by plotting %T against /xg P on semilog 
paper. 



1.037 



Determination 



Transfer aliquot (^15 mL contg <75 xtg P) of Sample Soln X to 
100 mL vol. flask. Add 5 mL NH 4 molybdate soln and mix. Add 
5 mL N 2 H 4 .H 2 S0 4 soln, dil. to 70 mL with H 2 0, and mix. Place 
flask in boiling H 2 9 min. Remove, cool rapidly, and dil. to vol. 
Use /xg P soln, 1.036, to set 100% T at 827 nm. Read %T for 
sample soln and det. fxg P from std curve. Calc. % P in sample. 



Titanium 



1.038 



Reagents 

(a) Titanium std soins. — {1) Stock soln. — 100 M9 Ti/mL. Place 
0.1668 g Ti0 2 and 2 g K 2 S 2 7 in Pt crucible. Heat covered crucible 
gently at first and then at dull red ca 15 min. Dissolve melt in 
50 mL H 2 S0 4 (1 + 1) and dil. to 1 L with H 2 0. (2) Working soln — 
5 /xg r i/mL Dil. 25 mL stock soln to 500 mL. 

(b) \cetate buffer soln. — pH 4.7. Dissolve 41 g anhyd. NaOAc 
in H 2 C add 30 mL HOAc, and dil. to 1 L. 

(c) Disodium - l,2-dihydroxybenzene-3, 5-dis uifonate ( 7 iron ) 
soln. — Dissolve 4 g Tiron in H 2 and dil. to 100 mL. 

1 .039 Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 5, 50, and 75 fxg Ti as in 
detn, but do not add dithionite to stds. Prep, std curve by plotting 
%T against /*g Ti on semilog paper. 



1.040 



Determination 



Transfer aliquot (<75 xig Ti) of Sample Soln X to 50 mL beaker. 
Dil. to ca 25 mL with H 2 0. Add 5 mL Tiron soln and then NH 4 OH 
(1+9) dropwise until soln is neut to Congo Red paper. (Tiron 



soln must be added before pH is adjusted.) Transfer to 50 mL 
vol. flask, add 5 mL buffer soln, dil. to vol. with H 2 0, and mix 
thoroly. Add 25 mg dithionite (Na 2 S 2 4 ) and dissolve with min. 
agitation (to avoid reappearance of blue). Use ttg Ti soln, 
1.039, to set 100% T at 410 nm. Read %T for sample soln within 
15 min after adding dithionite. Det. /xg Ti from std curve. Calc. 
% Ti in sample. 

Silicon 

(Clean all glassware with HCI (1 + 1).) 

1 .041 Reagents 

(a) Silicon std soln.— 20 /xg Si/mL. Place 0.0428 g pure Si0 2 in 
75 mL Ni crucible and treat as in 1.025(a), but dil. with H 2 to 
1 L instead of 100 mL. 

(b) Tartaric acid soln. — Dissolve 50 g tartaric acid in H 2 and 
dil. to 500 mL. Store in plastic bottle. 

(c) Ammonium molybdate soln. — Dissolve 7.5 g 
(NH 4 ) 6 Mo 7 24 .4H 2 in 75 mL H 2 0, add 10 mL H 2 S0 4 (1 + 1), and 
dil. to 100 mL with H 2 0. Store in plastic bottle. 

(d) Reducing soln.— Dissolve 0.7 g Na 2 S0 3 in 10 mL H 2 0. Add 
0.15 g 1-amino-2-naphthol-4-sulfonic acid and stir until dis- 
solved. Dissolve 9 g NaHS0 3 in 90 mL H 2 0, add to first soln, and 
mix. Store in plastic bottle. 

1 .042 Preparation of Standard Curve 

Treat aliquots of std soln contg 0, 20, 100, and 200 fxg Si as in 
detn. Prep, std curve by plotting %T against /xg Si on semilog 
paper. 



1.043 



Determination 



Transfer 10 mL Sample Soln Y to 100 mL vol. flask (use 
Sample Soln X for limestones contg <0.2% Si) and add 1 mL NH 4 
molybdate soln with swirling. Mix well, and let stand 10 min. 
Add 4 mL tartaric acid soln with swirling, and mix well. Add 1 
mL reducing soln with swirling, dil. to vol., mix well, and let 
stand ^30 min. Use fxg Si soln, 1.042, to set 100% T at 650 
nm. Read %T for sample soln and det. fxg Si from std curve. 
Calc. % Si in sample. 



SELECTED REFERENCES 

(7) JAOAC 7, 252(1924). 

(2) JAOAC 7, 252(1924); 55, 539(1972). 

(3) JAOAC 38, 240(1955). 

(4) Ind. Eng. Chem. 20, 312(1928); JAOAC 11, 153 (1928); 14, 
283(1931). 

(5) JAOAC 38, 413(1955). 

(6) JAOAC 27, 74, 532(1944); 28, 310(1945); 31, 71(1948). 

(7) JAOAC 31, 715(1948). 

(8) JAOAC 46, 603(1963); 47, 1019(1964). 

(9) U.S. Geol. Survey Bull. 700, p. 106; Ind. Eng. Chem. 9, 
1114(1917). 

(70) Washington, "Chemical Analysis of Rocks," 3rd Ed., 1919, 
p. 181. 

(11) JAOAC 45, 1(1962); 46, 611(1963); 50, 190(1967). 

(12) JAOAC 47, 1019(1964). 



2. Fertiliiers 



Sampling — Official Final Action 
2.001 Solid Fertilizers ( 7 ) 

(a) Bagged fertilizers. — Use slotted single or double tube trier 
with solid cone tip, constructed of stainless steel or brass, (Do 
not use unplated brass for samples on which micronutrients are 
to be detd.) Trier length, exclusive of handle, should be approx. 
length of filled bag to be sampled, but >25"; length of slot, 
>23"; width of slot 3*0.5"; and id =* 5 / 8 ". 

Take sample as follows: Lay bag horizontally and remove core 
diagonally from end to end. From lots of ^10 bags, take core 
from each of 10 bags. When necessary to sample lots of <10 
bags, take 10 cores but at least 1 core from each bag present. 
For small packages (^10 lb), take 1 entire package as sample. 

(b) Bulk fertilizers, including railroad car-size lots. — Use trier 
of design represented in Table 2:01. 



Table 2:01 


Trier specifications 










Length, in. 


od, in. 


id, in. 


Compartments 


Trier 


No. 


Size, in. 


Missouri 
552 Grain 3 
Missouri "D' 


59 
63 

'* 52 


1% 
1 3 / 8 
1% 


Vb 
V/s 
1 


8 

11 
1 


3 

VA 
45 



Triers available from: 

a Seedboro Equipment Co., 618 W Jackson Blvd, Chicago, IL 60606. 
b American Tool and Die, Inc., 1105 Maple St, West Des Moines, lA 
50265. 

Draw 10 vertical cores distributed in std concentric sampling 
pattern (Fig. 2:01) of such design that each core represents 
approx. equal fractions of lot. 

Bulk shipments may be sampled at time of loading or un- 
loading by passing sampling cup, Fig. 2:02 (mouth dimensions: 
width %", length 16" or as long as max. diam. of stream), thru 
entire stream of material as it drops from belt or chute. Make 



V^x 8 


(3 2) 5) 


10 v *-i--' 9 



FIG. 2:01— Sampling pattern 



OPEN 
MOUTH 




sampling such as to assure 5=10 equal-timed-spaced passes 
thruout transfer operation. Stream samples are not applicable 
unless uniform continuous flow of fertilizer is maintained for 
>3 min while lot is being sampled. 

(c) Preparation of sample.—Place composite sample in air- 
tight container and deliver entire sample to laboratory. Reduce 
composite sample in laboratory, using riffle. 

2.002 Liquid Fertilizers (2) 

{In Absence of Free Ammonia) 

(a) Clear solns. — (Mixed liqs and N solns.) Secure sample 
directly from mixing vat, storage tank, or delivery tank after 
thoro mixing. Take sample from surface or thru direct tap. Flush 
direct tap, or delivery line and faucet, and collect sample in glass 
or polyethylene container. Alternatively, lower sample container 
into well mixed material thru port in top of tank and let fill. Seal 
container tightly. 

(b) Fluid fertilizers with suspended materia!. — (Salt suspen- 
sions and slurries.) Agitate material in storage until thoroly 
mixed (15 min usually adequate) before taking sample. Sample 
directly as in (a), or use 500 mL Missouri or Indiana sampling 
bottle, Fig. 2:03. Lower sampling bottle from top opening to 
bottom of tank and raise slowly while filling. Transfer to sample 
bottle and seal tightly. 

Alternatively, secure sample from tap on recirculation line 
after agitating and recirculating simultaneously until thoroly 
mixed. Draw sample while recirculating. If recirculation line is 



^ Air escape hole 1/8" 

— Fluid Intake tube: 1/4" for solutions 

3/8" for suspensions 
and slurries 




Missouri 



RG. 2:02— Sampling cup 



Air escape hole 3mm 
Fluid intake tube 7mm - 
Stainless steel jacket- 



FIG. 2:03 — Missouri and Indiana weighted restricted-fill fluid fertilizer 

sampling bottles designed to fill while being lowered (and raised) in 

storage tanks 



2. Fertilizers 



AOAC Methods (1980) 



attached to manifold delivery line, allowing cross-contamination, 
pump ca 30 cm (V) or 2000 L (500 gal.) into temporary storage 
tank, then sample from recirculation line as above or from 
delivery line. Transfer to sample bottle and seal tightly. 



time.) Close sample valve, remove sample tube, partially col- 
lapse bottle, and cap tightly. Reweigh (±0.1 g) and calc. wt 
sample. Cool to 20°, transfer to 1 or 2 L vol. flask, dil. to vol. with 
H 2 0, mix thoroly, and take aliquots for analysis. 



Ammoniacal Solutions [3) 

2.003 Apparatus 

(a) Container. — Polyethylene reagent-form bottle with but- 
tress-type cap, 1 L (1 qt) capacity. 

(b) Sample flow control apparatus. — Construct from following 
fittings: IV2 x %" reducing bushing; %" tee; V*" nipple 12-18" 
long (length not critical); two %" stainless steel, blunt-nose 
needle valves with hose connections (Hoke No. 328; Hoke Inc., 
1 Tenakill Pk, Cresskill, NJ 07626). All fittings except valves can 
be either Al or stainless steel. (See Fig. 2:04.) 

Attach valves directly to tee which is then attached to reducing 
bushing thru nipple. To both valves attach !4" id Tygon tubing 
{Hoke No. 314A hose connection), 12" length to sample valve 
and sufficient length to vent valve to reach disposal area or 
container. To free end of sample tubing attach 3" length of 14" 
glass or stainless steet tubing inserted thru No. 4 rubber stopper. 
To exit end of metal tube attach addnl 6" length of Tygon tubing. 
Make certain all connections are tight. App. can be attached 
directly to tank cars, but requires addnl coupling, which varies 
with installation, to attach to storage tanks. 1 Y 2 " "quick coupler" 
(Ever-Tite Coupling Co., 254 W 54th St, New York, NY 10019) 
suffices in most cases. 

2.004 Sampling 

Prep, sample bottle in laboratory by adding ca 500 mL H 2 0, 
replacing cap, and weighing accurately (±0.1 g). Attach sampling 
app. to car or tank and, with sample valve closed, flush line thru 
vent valve. Partially collapse sample bottle, insert sample tube 
with stopper, and seat tightly. With sample tube dipping below 
surface of H 2 in bottle, throttle vent valve to maintain small 
flow of soln and partially open sample valve, collecting ca 100 
mL sample. (Bottle should not expand to full size during this 



■ NEEDLE VALVE - BLUNT 
NEEDLE 




U 



COUPLER 



Anhydrous Ammonia (3) 

(Caution; Use extreme care in handling anhyd. NH 3 . Suitable gas 
mask and rubber gloves are required. See 51.032.) 

2.005 Sampling 

Use sample tube of thermal shock-resistant glass calibrated 
to contain 100 mL and graduated in 0.05 mL subdivisions up to 
0.5 mL. (Dupont special oil centrf. tube or ASTM long-form oil 
tube is satisfactory.) Flush line and fill tube to 100 mL mark with 
sample in such manner that condensing moisture will not enter 
sample tube. (Skirt attached to end of sample line will drain 
moisture away.) 

2.006 Water and Nitrogen 

Immediately close sample tube with tight-fitting rubber stop- 
per into which is inserted tight-fitting piece of 6 mm id glass 
tubing 5-8 cm long, bent at its exit from outer end of stopper 
to let gases escape but to exclude entrance of moisture or 
moisture-laden air. Place in H 2 bath at approx. air temp, and 
let NH 3 evap. When temp, of sample tube is ca that of bath, 
remove tube, wipe outer surface, and det. vol. of residue. 

% H 2 in sample = mL residue x C, 
where C = 0.74, 0.70, or 0.66 for pressures in original containers 
of 100, 150, or 200 psi, resp. 

% N = (100 - % H 2 0) x 0.8224. 



2.007 Preparation of Sample (4) — Official Final Action 

Reduce gross sample to amt sufficient for analysis or grind 
3=225 g (0.5 lb) of reduced sample without previous sieving. For 
fertilizer materials and moist fertilizer mixts, grind to pass sieve 
with 1 mm circular openings, or No. 20 sieve; for dry mixts that 
tend to segregate, grind to pass No. 40 sieve. Grind as rapidly 
as possible to avoid loss or gain of moisture during operation. 
Mix thoroly and store in tightly stoppered bottles. 

2.008 * Mechanical Analysis of Bone, Tankage, and * 

Basic Slag (5)— Official Final Action 

Sieve thru circular openings 0.5 mm diam. See 2.008, 1 1th ed. 

Mechanical Analysis of Phosphate Rock {6) 
Official Final Action 

2.009 Apparatus 

(a) Water pressure control. — See Fig. 2:05. Connect valve, A 
std pressure gage, B, and aerator, C, with W diam. pipe. 

(b) Sieves. — Nos. 100 and 200, bronze or stainless steel cloth, 
checked against certified sieves. Sieves 8" diam. and 2" in depth 
to sieve cloth are recommended for both wet and dry sieving, 
but other sizes may be used if detd to be suitable under 
conditions of method. (Other sieves in U.S. series may be used, 
with precaution to ensure complete sepn of sample into desired 
fractions.) 

(c) Sieve shaker.— Ro-Tap (W. S. Tyler, Inc., 8200 Tyler Blvd, 
Mentor, OH 44060), Syntron (FMC Corp., Material Handling 



FIG. 2:04 — Sampling apparatus for ammoniacal solutions, including 
"quick coupler" for attaching to storage tanks 



* Surplus method— see inside front cover. 



AOAC Methods (1980) 



Phosphorus 




FIG. 2:05 — Apparatus for control of water pressure 

Equipment Div., Homer City, PA 15748), or other suitable ma- 
chine. 

2.010 Reagent 

Dispersing agent — Dissolve 36 g Na hexam eta phosphate and 
8 g Na 2 C0 3 in H 2 and dil. to 1 L 



2.011 



Determination 



(a) Ground phosphate rock. — Place 100 g sample on No. 200 
sieve and wash with moderate stream of tap H 2 at max. gage 
pressure of 0.28 kg/sq cm (4 Ib/sq in.) until H 2 passing sieve 
is ciear, with care to avoid loss of sample by splashing. Dry 
material remaining on sieve at 105° and transfer to No. 100 sieve 
in series with No. 200 sieve of same diam. and depth. Shake 8 
min in mech. shaker. Det. % sample passing No. 100 sieve by 
subtracting wt of material retained on that sieve from 100. Det. 
% sample passing No. 200 sieve by subtracting sum of wts of 
material retained on that sieve and on No. 100 sieve from 100. 

(b) Soft phosphate with colloidal clay. — Add 100 g sample to 
rapidly stirred soln of 50 mL dispersing agent and 450 ml_ tap 
H 2 0, with care to avoid contact of unwetted material with shaft 
of stirrer and side of beaker. Stir 5 min after addn of sample is 
completed. Transfer slurry to No. 200 sieve and proceed as in 
(a). 

2.012 Total Water— Official Final Action 

(Not applicable to samples that yield volatile 
substances other than H 2 at drying temp.) 

Heat 2 g sample, 2.007, 5 hr in oven at 100±1°. In case of 
NaN0 3/ (NH 4 ) 2 S0 4 , and K salts, heat to const wt at 130±1°. Report 
% loss in wt as H 2 at temp. used. 

FREE WATER 
Vacuum-Desiccation Methods (7) 

2.013 Method I— Official Final Action 

Place 2 g prepd sample, 2.007, in tared weighing dish. (Weigh 
extremely hygroscopic or damp materials by difference in 
covered dishes.) Dry sample at 25-30° (precise results depend 
on as const a temp, as possible) in vac. desiccator over anhyd. 



Mg(CI0 4 ) 2 , P 2 5 , or BaO, under ^50 cm (20") or =£55 cm (22") 
vac. (20-25 cm (8-10") absolute pressure) 16-18 hr. Reweigh, 
and report % loss in wt as free H 2 0. 

2.014 Method II— Official Final Action 

(Not applicable to samples which yield volatile 
substances other than H 2 0) 

Weigh 2 g prepd sample, 2.007, into tared glass weighing 
dish. Dry sample 2 hr ±10 min at 50±1.5° in oven under vac. of 
48-53 cm (19-21") (23-28 cm (9-1 1") absolute pressure). (Temp, 
control within specified limits thruout oven chamber is essential.) 
Maintain vac. by passing desiccated air thru chamber. Cool 
dried sample in desiccator and reweigh. Report % loss in wt as 
free H 2 0. 

Alternative Extraction Method [8) 
Official Final Action 

2.015 Principle 

Free H 2 is extd with dioxane and detd by titrn with Karl 
Fischer reagent. 

2.016 Reagents 

(Keep exposure of org. reagents to air at min.) 

(a) Karl Fischer reagent — Stabilized single soln (Fisher Scien- 
tific Co., So-K-3, or equiv.) dild ca 1 + 1 with stabilized diluent 
{Fisher, So-K-5, or equiv.), or soln equiv. to 2.5 mg H 2 0/mL. Stdze 
daily with ca 0.2 g Na tartrate.2H 2 0. 1 mg Na tartrate.2H 2 = 
0.1566 mg H 2 0. 

(b) Methanol.— Low in H 2 0. 



2.017 



Determination 



Accurately weigh 2.5 g prepd sample, 2.007, into 125 mL 
erlenmeyer, add 50.0 mL 1,4-dioxane, stopper, mix by swirling, 
and let stand 15 min. Mix thoroly by swirling, and centrf. in 
closed tube. {Caution: See 51.005.) 

Transfer 10 mL aliquot to titrn vessel contg pretitrd MeOH 
and titr. with Karl Fischer reagent. (Discard contents of titrn 
vessel after 3 titrns, replace with enough MeOH to cover 
electrodes, and pretitr. before proceeding with next sample.) 
Det. blank on 10 mL dioxane as above and subtract from sample 
detns. Calc. and report as free H 2 0. 

2.018 * Acid-Insoluble Ash [9) * 
Official Final Action 

HCI (1+4) digestion at 100°, ignition at 800°, and redigestion. 
See 2.015, 11th ed. 

PHOSPHORUS 
Total Phosphorus 



2.019 



Reagent 



Magnesium nitrate soln. — Dissolve 950 g P-free Mg(N0 3 ) 2 . 
6H 2 in H 2 and dil. to 1 L. 

2.020 Preparation of Solution — Official Final Action 

(Caution: See 51.019, 51.026, 51.028, 51.030, and 51.069.) 

Treat 1 g sample by (a), (b), (c), (d), or (e), as indicated. Cool 
soln, transfer to 200 or 250 mL vol. flask, dil. to vol., mix, and 
filter thru dry filter. 

* Surplus method— see inside front cover. 



10 



2. Fertilizers 



AOAC Methods (1980) 



(a) Materials containing small quantities of organic matter. — 
Dissolve in 30 mL HN0 3 and 3-5 mL HCI, and boil until org. 
matter is destroyed (30 min for liqs and suspensions). 

*(b) Fertilizers containing much Fe or Al phosphate, and 
basic slag.*— Dissolve in 15-30 mL HCI and 3-10 mL HN0 3 . 

(c) Organic material like cottonseed meal alone or in mix- 
tures. — Eva p. with 5 mL Mg(N0 3 ) 2 soln, 2.019, ignite, and dissolve 
in HCI. 

*(d} Materials or mixtures containing large amounts of or- 
ganic matter. if — Digestion with H 2 S0 4 and NaN0 3 or KN0 3 . See 
2.017(d), 11th ed. 

(e) All fertilizers.— Boil gently 30-45 min with 20-30 mL HN0 3 
in suitable flask (preferably Kjeldahl for samples contg large 
amts of org. matter) to oxidize all easily oxidizable matter. Cool. 
Add 10-20 mL 70-72% HCI0 4 . Boil very gently until soln is 
colorless or nearly so and dense white fumes appear in flask. 
Do not boil to dryness at any time (Danger!). (With samples 
contg large amts of org. matter, raise temp, tc fuming point, ca 
170°, over period of s=1 hr.) Cool slightly, add 50 mL H 2 0, and 
boil few min. 



Spectrophotometry Molybdovanadophosphate 
Method ( 10)— Official Final Action 

(Not applicable to materials yielding colored solns or solns 
contg ions other than orthophosphate which form colored 
complexes with molybdovanadate. Not recommended for 

basic slag.) 
2.021 Apparatus 

Photometer. — Beckman Instruments, Inc. Model DU {current 
models 24/25) spectrophtr with stray light filter and matched 1 
cm cells. With other photometers analyst must det. suitability 
for use and conditions for satisfactory performance. Means for 
dispelling heat from light source is desirable. 



2.022 



Reagents 



(a) Molybdovanadate reagent. — Dissolve 40 g NH 4 molyb- 
date.4H 2 in 400 mL hot H 2 and cool. Dissolve 2 g NH 4 
metavanadate in 250 mL hot H 2 0, cool, and add 450 mL 70% 
HCI0 4 . [Caution: See 51.028(a) and (d).) Gradually add molybdate 
soln to vanadate soln with stirring, and dil. to 2 L. 

(b) Phosphate std soln.— Dry pure KH 2 P0 4 (52.15% P 2 5 ) 2 hr 
at 105°. Prep, solns contg 0.4-1.0 mg P 2 5 /mL in 0.1 mg 
increments by weighing 0.0767, 0.0959, 0.1151, 0.1342, 0.1534, 
0,1726, and 0.1918 g KH 2 P0 4 and dilg each to 100 mL with H 2 0. 
Prep, fresh solns contg 0.4 and 0.7 mg P 2 5 /mL weekly. 

2.023 Preparation of Standard Curve 

Pipet 5 mL aliquots of 7 std phosphate solns (2-5 mg P 2 5 / 
aliquot) into 100 mL vol. flasks and add 45 mL H 2 0. Then, within 
5 min for entire series, add 20 mL molybdovanadate reagent by 
buret or pipet, dil. to vol. and mix. Let stand 10 min. 

Select 2 absorption cells (std and sample cells) and fill both 
with 2 mg std. Set spectrophtr to 400 nm and adjust to zero A 
with std cell. Sample cell must check zero A within 0.001 unit; 
otherwise read A for sample cell and correct subsequent read- 
ings. (Choose cell showing pos. A against other as sample cell 
so that this pos.>4 is always subtracted.) Using sample cell, det. 
A of other stds with instrument adjusted to zero>4 for 2 mg std. 
After each detn empty and refill cell contg 2 mg std, and readjust 
zero to avoid error that might arise from temp, changes. Plot>4 
against concn in mg P 2 5 /mL std soln. 



^ Surplus method — see inside front cover. 



2.024 Preparation of Solution 

Treat 1 g sample as in 2.020, preferably (e), when these acids 
are suitable solv. (Soln should be free of N oxides and NOCL) 

(a) For P 2 5 content ^5%, dil. to 250 mL 

(b) For P 2 5 content >5%, dil. to such vol. that 5 or 10 mL 
aliquot contains 2-5 mg P 2 5 . 

2.025 Determination 

Pipet, into 100 mL vol. flasks, 5 mL aliquots of std phosphate 
solns contg 2 and 3.5 mg P 2 5 /aliquot, resp., and develop color 
as in 2.023. Adjust instrument to zero>4 for 2 mg std, and det. 
A of 3.5 mg std. (It is essential that/4 of latter std be practically 
identical with corresponding value on std curve.) 

(a) Samples containing up to 5% P 2 O s — Pipet, into 100 mL 
vol. flask, 5 mL sample soln, 2.024(a), and 5 mL std phosphate 
soln contg 2 mg P 2 5 . Develop color and det. A concurrently 
with and in same manner as for std phosphate solns in preceding 
par., with instrument adjusted to zero>4 for 2 mg std. Read P 2 5 
concn from std curve. With series of sample solns, empty and 
refill cell contg 2 mg std after each detn. 

% P 2 5 in sample = 100 x [(mg P 2 5 from std curve - 2)/20]. 

(b) Samples containing more than 5% P 2 5 . — Pipet 5 or 10 
mL sample soln, 2.024(b), into 100 mL vol. flask. Without adding 
std phosphate soln, proceed as in (a). 

% P 2 5 in sample = 100 x (mg P 2 5 from std 

curve/mg sample in aliquot). 



Gravimetric Quino/inium Molybdophosphate 
Method ( 77) — -Official Final Action 



2.026 



Reagents 



(Store solns in polyethylene bottles.) 



(a) Citric-molybdic acid reagent — Dissolve 54 g 100% mo- 
lybdic anhydride (Mo0 3 ) and 12 g NaOH with stirring in 400 mL 
hot H 2 0, and cool. Dissolve 60 g citric acid in mixt. of 140 mL 
HCI and 200 mL H 2 0, and cool. Gradually add molybdic soln to 
citric acid soln with stirring. Cool, filter, and dil. to 1 L. (Soln may 
be green or blue; color deepens on exposure to light.) If 
necessary, add 0.5% KBr0 3 soln dropwtse until green color 
pales. Store in dark. 

(b) Quinoline soln.- Dissolve 50 mL synthetic quinoline, with 
stirring, in mixt. of 60 mL HCI and 300 mL H 2 0. Cool, dil. to 1 L, 
and filter. 

(c) Quimoclac reagent. — Dissolve 70 g Na molybdate.2H 2 in 
150 mL H 2 0. Dissolve 60 g citric acid in mixt. of 85 mL HN0 3 and 
150 mL H 2 0, and cool. Gradually add molybdate soln to citric 
acid-HN0 3 mixt. with stirring. Dissolve 5 mL synthetic quinoline 
in mixt. of 35 mL HN0 3 and 100 mL H 2 0. Gradually add this soln 
to molybdate-citric acid-HN0 3 soln, mix, and let stand 24 hr. 
Filter, add 280 mL acetone, dil. to 1 L with H 2 0, and mix. 

2.027 Preparation of Solution 

Treat 1 g sample as in 2.020, dilg to 200 mL. 



2.028 



Determination 



Pipet, into 500 mL erlenmeyer, aliquot contg =£25 mg P 2 5 
and dil. to ca 100 mL with H 2 0. Continue by one of the following 
methods: 

(a) Add 30 mL citric-molybdic acid reagent and boil gently 3 
min. (Soln must be ppt-free at this time.) Remove from heat and 
swirl carefully. Immediately add 10 mL quinoline soln from buret 
with continuous swirling. (Add first 3-4 mL dropwise and 
remainder in steady stream.) Or: 



AOAC Methods (1980) 



Phosphorus 



11 



(b) Add 50 mL quimociac reagent, cover with watch glass, 
place on hot plate in well-ventilated hood, and boil 1 min. 

After treatment by (a) or (b), cool to room temp., swirl carefully 
3-4 times during cooling, filter into gooch with glass fiber filter 
paper previously dried at 250° and weighed, and wash with five 
25 mL portions of H 2 0. Dry crucible and contents 30 min at 250°, 
cool in desiccator to room temp., and weigh as (C 9 H 7 N)3H 3 [P04. 
12Mo0 3 ]. Subtract wt reagent blank. Multiply by 0.03207 to 
obtain wt P 2 O s (or by 0.01400 for P). Report as % P 2 5 (or % P). 

Alkalimetric Quinolinium Molybdophosphate 
Method {12)— Official Final Action 

2.029 Reagents 

(a) Quimociac reagent — See 2.026(c). 

(b) Sodium hydroxide std soln.— (1 mL = 1 mg P 2 5 .) Dil. 
366.32 mL 1A/ NaOH, 50.032-^50.036, to 1 L with H 2 0. 

(c) Nitric acid std soln. — Prep. HN0 3 soln equiv. to concn of 
(b) and stdze by titrg against (b), using phthln. (For greater 
precision, use HN0 3 soln corresponding to 1/5 concn of (b).) 

(d) Citric acid.— 10% (w/v). 

(e) Indicators.— (1) Thymol blue soln.— 0.1%. Add 2.2 mL0.1A/ 
NaOH to 0.1 g thymol blue and dil. to 100 mL with 50% alcohol. 
(2) Phenolphthalein. — 0.1%. Dissolve 0.1 g phthln in 100 mL 
50% alcohol. {3) Mixed indicator.— -Mix 3 vols (/) and 2 vols (2). 

2.030 Preparation of Sample Solution 

Treat 1 g sample as in 2.020, first par. and (a) or (e). 



2.031 



Determination 



(a) Precipitation. — Transfer aliquot contg =e30 mg P 2 5 and 
*s5 mL coned acid to 500 mL erienmeyer, add 20 mL citric acid 
soln, and adjust to ca 100 mL. Add 60 mL quimociac reagent, 
immediately cover with watch glass, and place on medium 
temp, hot plate. After soln comes to bp, move to cooler portion 
of hot plate and boil gently 1 min. Let cool until flask can be 
handled comfortably with bare hand. 

(b) Filtration and washing. — Prep, pulped- pa per pad ca 6 mm 
thick on perforated porcelain disk in funnel by adding 5*2 approx. 
equal increments of H 2 suspension of pulped paper and sucking 
dry with vac. between addns. Swirl flask, pour contents onto 
filter, and wash flask with five ca 15 mL portions H 2 0, adding 
washings to funnel. Immediately after funnel has emptied, wash 
down sides with ca 15 mL H 2 to remove residual acetone, 
which causes excessively fast drying and later lump formation 
if allowed to evap. Wash with 3 addnl 15 mL portions H 2 0, 
letting funnel empty between addns. Keep drying of ppt to min. 
Using only jet of H 2 0, transfer ppt and pad to pptn flask and 
break up pad with jet of H 2 0. Do not smear ppt against funnel 
or flask. 

(c) Titration. — Titr. with std NaOH soln and add 3-5 mL 
excess. Add 1 mL mixed indicator and titr. with std HN0 3 soln 
to grey-blue end point. If overtitrd (greenish-yellow), add addnl 
excess std NaOH soln and titr. to grey-blue. 

(d) Blank. — Det. blank on all reagents, adding known amt (1-2 
mg) of P 2 5 . Use 1 + 9 dilns of std NaOH and HN0 3 for titrn and 
subtract theoretical titer equiv. to P 2 5 added from experimental 
titer. Calc. difference equiv. to 0.3663A/ NaOH and subtract this 
blank from all sample detns. 

Calc. and report as % P 2 5 . 

Automated Method [13) — Official First Action 
2.032 Principle 

Samples are extd for direct available P 2 5 or for total P 2 5 



detns. Destruction of coloring matter, hydrolysis of nonortho- 
phosphates, and elimination of citrate effect are accomplished 
by digestion with 4A/ HCI0 4 at 95°. Digested samples are reacted 
with molybdovanadate reagent, and A of resulting complex is 
read in flowcell at 420 nm in range 0,15-0.35 mg P 2 5 /mL. 

2.033 Apparatus and Reagents 

(Caution: See 51,028.) 

(a) Automatic analyzer.— AutoAnalyzer with following mod- 
ules (Technicon Instruments Corp., or equiv.): Sampler II or IV 
with 40/hr (4:1) cam; proportioning pump III; P 2 5 anal, cartridge 
(with 2 heating baths, each contg 10.6 mL coil held at 95±1°; or 
AAI type heating bath contg one 40' x 1.6 mm id coil and 
holding constant temp, of 95±1°); AAII single channel color- 
imeter with 15 x 1.5 or 2.0 mm id flowcell and 420 nm 
interference filters; voltage stabilizer; and recorder. Construct 
manifold as in flow diagram, Fig. 2:06. 

(b) Molybdovanadate reagent— Dissolve 16.5 g NH 4 molyb- 
date.4H 2 in 400 mL hot H 2 0, and cool. Dissolve 0.6 g NH 4 
metavanadate in 250 mL hot H 2 0, cool, and add 60 mL 70% 
HCIO4. Gradually add molybdate soln to vanadate soln with 
stirring. Add 2 mL wetting agent, (e), and dil. to 2 L. 

(c) Perchloric acid.-AN. Add 342 mL 70% HCI0 4 to 500 mL 
H 2 in 1 L vol. flask. Add 1 mL wetting agent, and dil. to vol. 

(d) Sampler wash soln. — Add 1 mL wetting agent to 1 L H 2 0, 
and mix well. 

(e) Wetting a^enf.— Ultrawet 60 L (Technicon No. T01-0214), 
or equiv. 

(f) Phosphorus std solns. — [1) Stock soln. — 10 mg P 2 5 /mL. 
Dissolve 9.5880 g dried (2 hr at 1 05°) KH 2 P0 4 primary std (52. 1 5% 
P 2 5 ) in H 2 0, and dil. to 500 mL with H 2 0. {2) Working solns — 
0.15, 0.19, 0.23, 0.27, 0.31, and 0.35 mg P 2 5 /mL. Using 25 mL 
buret, accurately measure 7.5, 9.5, 11.5, 13.5, 15.5, and 17.5 mL 
stock soln into six 500 mL vol, flasks. Dil. each to vol. with H 2 0, 
and mix. (3) Working soln for samples ^7% P 2 G 5 — 2 mg 
P 2 5 /mL Pipet 100 mL stock soln into 500 mL vol. flask, dil. to 
vol. with H 2 0, and mix. 



2.034 



Preparation of Samples 



Prep, samples for direct available P 2 5 detn as in 2.050(a). 
Prep, samples for total P 2 5 detn as in 2.020(a) or (e), and dil. 
to 250 mL 

2.035 Analytical System 

(Technicon part numbers are given to aid in construction of 
manifold; equiv. coils, fittings, etc., are satisfactory.) 

Sample, air, and 4/V HCI0 4 are combined thru injection fitting 
(No. 116-0489) and mixed in 20T coil (No. 157-0248). Stream 
proceeds to heating bath(s) before resample thru modified AO 
fitting. Resample, air, and molybdovanadate reagent are com- 
bined thru injection fitting (No. 116-0489). Mixing and color 
development takes place in two 20T coils (No. 1 57-0248) before 
measurement at 420 nm. If only total P 2 O s samples are to be 
analyzed, heating bath can be removed and 4/V HCI0 4 soln 
replaced by sampler wash soln, (d). Heating bath(s) and acid 
soln are necessary only when analyzing samples for direct 
available P 2 5 or combination of direct available and total P 2 5 
detns. 

If manifold is to be constructed following flow diagram, use 
clear std pump tubes for all air and soln flows. All fittings, coils, 
and glass transmission lines are AAII type and size. Use 1.6 mm 
glass transmission tubing for all connections after pump to 
colorimeter. Construct modified AO fitting, following heating 
bath, by using AO fitting, N13 stainless steel nipple connector, 



12 



2. Fertilizers 



AOAC Methods (1980) 



SAMPLER II 



20T 
MIXER; 



INJECTION 
FITTING 



FLOW,ml/Min 




\ °- 32 
\l 0.60 



AIR 



40' COIL 
1.6 mm 1.0. 



HCI04 



0.23 



SAMPLE 




HEATING BATH 



INJECTION 
FITTING 




0.32 



AIR 



1.00 MO-V REAGENT 



0.23 DIG. SAMPLE 



20 T 
MIXER 



MODIFIED AO 



7 



WASTE - 



0.60 



Dl° 



SAMPLER II- 
WASTE ^ 



2.00 



H20 



0.80 



FLOWCELL 




iT 

COLORIMETER RECORDER 

15 mm FLOWCELL 
420 nm FILTERS 



6 FT. LENGTH 0.030 1.D-' 
TRANSMISSION TUBING 



WASTE 



POSITIONED WITH CAPILLARY SIDEARM 
ON BOTTOM 



FIG. 2:06 — Flow diagram for automated analysis for phosphorus 



and 1.3 cm length of 0.035" id Tygon tubing. Insert N13 nipple 
approx. halfway into 0.035" Tygon tubing. Insert tubing into 
side arm of AO fitting far enough so resample line will not pump 
any air. Connect D1 fitting directly to waste side of AO fitting; 
position D1 fitting with capillary side arm on bottom. Attach 0.6 
mL/min pump tube to top arm of D1 fitting, and attach 1.8 m 
(6') of 0.030" id transmission tubing to bottom arm. All air 
segments must pass thru 0.6 mL/min tube, leaving continuous 
column of liq. in 1.8 m length of tubing to provide for constant 
back pressure on heating bath coil. Length of resample pump 
tube should be ^2.5 cm from shoulder at entrance end. 

2.036 Start-Up 

Start automatic system, place all lines in resp. solns, and let 
equilibrate ^30 min. Proceed as in 2.038. 



2.037 



Shut-Down 



Pump water thru reagent lines >30 min. Do not remove HCI0 4 
lines from reagent until 20 min after last sample is run. 



2.038 



Check and Calibration 



After equilibration, set colorimeter to damp 1 position and 
pump 0.15 mg P 2 5 /mL working std soln continuously thru 
system. Adjust colorimeter baseline to read 10% of full scale. 
Pump 0.35 mg P 2 5 /mL std and adjust std calibration to read 
90% of full scale. Range of 0.15-0.35 mg P 2 5 /rnL will expand 
to read 10-90% of full scale. Check of bubble flow pattern will 
give indication of performance of system. Perfect bubble pattern 
is required to obtain optimum peak shapes. Check for air bubble 
in flowcell if noisy conditions exist. To check system carryover, 



place three 0.35 mg/mL stds, followed by three 0.15 mg/mL 
stds thru system. If first 0.15 mg/mL std following 0.35 mg/mL 
std is 3=1 chart division higher than other 2, carryover is 
indicated. If carryover occurs, check entire system for poor 
connections. 



2.039 



Determination 



Pipet aliquot of sample soln {see Table 2:02) into 100 mL vol. 
flask, dil. to vol. with H 2 0, and mix by inversion 20 times. For 
sample contg <?% P 2 5 , pipet 10 mL working soln, (f)(3), into 
flask before diln. Place 0.15-0.35 mg P 2 5 /mL working std solns 
in tray in increasing order of concn, followed by group of 
samples. Analyze lowest concn std in duplicate, discarding first 
peak. Precede and follow each group of samples with std ref. 
curve to correct for possible drift. If drift between first and last 
set of stds is ^2 chart divisions, repeat sample analysis. Prep, 
std curve by averaging peak hts of first and second set of stds. 
Plot av. peak ht of stds against mg P 2 5 /mL contained in each 
std. Read mg P 2 5 /mL for each sample from graph. 
% P 2 5 = mg P 2 5 /mL from graph (-0.20, if spiked) xfx 100, 
where F = factor from Table 2.02. 



Table 2:02 Standard Dilutions 



% P 2 5 
Expected 



Aliquot (mL) 
Direct Available Total 



Factor 



1-7 


50 + "spike" 


25 + 


"spike" 


1 


8-16 


no diln 


50 




0.5 


17-34 


50 


25 




2 for direct available 


s*35 


25 


15 




1.667 for total 



AOAC Methods (1980) 



Phosphorus 



13 



Water-Soluble Phosphorus 

2.040 Preparation of Solution — Official Final Action 

Place 1 g sample on 9 cm filter and wash with small portions 
H 2 until filtrate measures ca 250 ml_. Add H 2 in fine stream 
directed around entire periphery of filter paper in circular path, 
ensuring that H 2 and solids are thoroly mixed with each addn. 
Let each portion pass thru filter before adding more and use 
suction if washing would not otherwise be complete within 1 
hr. If filtrate is turbid, add 1-2 mL HN0 3 , dil. to 250 mL, and mix. 

2.041 Gravimetric Quinolinium Molybdophosphate 
Method — Official Final Action 

Pipet aliquot contg ^25 mg P 2 5 into 500 mL erlenmeyer. Dil., 
if necessary, to 50 mL, add 10 mL HN0 3 (1 + 1), and boil gently 
10 min. Cool, dil. to 100 mL, and proceed as in 2.028(b). 

2.042 Alkalimetric Quinolinium Molybdophosphate 
Method — Official Final Action 

Pipet aliquot contg ^30 mg P 2 5 into 500 mL erlenmeyer. Dil., 
if necessary, to 50 mL, add 10 mL HN0 3 (1 + 1), boil gently 10 
min, cool, and proceed as in 2.031(a), beginning ". . . add 20 mL 
citric acid soln . . ." 

2.043 Spectrophotometric Molybdovanadophosphate 
Method — Official Final Action 

Adjust concn according to 2.024(a) or (b) and proceed as in 
2.025. 



Citrate-Insoluble Phosphorus [14) 
Official Final Action 

2.044 Reagents 

(a) Ammonium citrate soln. — Should have sp gr of 1 .09 at 20° 
and pH of 7.0 as detd potentiometrically. 

Dissolve 370 g cryst. citric acid in 1.5 L H 2 and nearly neutze 
by adding 345 mL NH 4 OH (28-29% NH 3 ). If concn of NH 3 is 
<28%, add correspondingly larger vol. and dissolve citric acid 
in correspondingly smaller vol. H 2 0. Cool, and check pH. Adjust 
with NH 4 0H (1+7) or citric acid soln to pH 7. Dil. soln, if 
necessary, to sp gr of 1.09 at 20°. (Vol. will be ca 2 L) Keep in 
tightly stoppered bottles and check pH from time to time. If pH 
has changed from 7.0, readjust. 

(b) Other reagents and solns.-^See 2.019, 2.022, or 2.026. 

2.045 Preparation of Extract 

(a) Acidulated samples, mixed fertilizers, and materials con- 
taining water-soluble compounds. — After removing H 2 0-sol. 
P 2 5 , 2.040, transfer filter and residue, within 1 hr, to 200 or 250 
mL flask contg 100 mL NH 4 citrate soln previously heated to 65°. 
Close flask tightly with smooth rubber stopper, shake vigorously 
until paper is reduced to pulp, and relieve pressure by removing 
stopper momentarily. Continuously agitate stoppered flask in 
const temp. app. at exactly 65°. (Action of app. should be such 
that dispersion of sample in citrate soln is continually maintained 
and entire inner surface of flask and stopper is continually 
bathed with soln.) 

Exactly 1 hr after adding filter and residue, remove flask from 
app. and immediately filter by suction as rapidly as possible 
thru Whatman No. 5 paper, or equiv., using buchner or ordinary 
funnel with Pt or other cone. Wash with H 2 at 65° until vol. 
filtrate is ca 350 mL, allowing time for thoro draining before 
adding more H 2 0. If material yields cloudy filtrate, wash with 



5% NH 4 N0 3 soln. Prep, citrate-insol. residue for analysis by one 
of following methods: 

(/) Dry paper and contents, transfer to crucible, ignite until 
all org. matter is destroyed, and digest with 10-15 mL HCI until 
all phosphate dissolves; or (2) treat wet filter and contents as 
in 2.020(a), (c), (d), or (e). Dil. soln to 250 mL, or other suitable 
vol., mix well, and filter thru dry paper. 

(b) Nonacidulated samples.— Place 1 g sample (ground to 
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm 
paper. Without previous washing with H 2 0, proceed as in (a). 
If sample contains much org. matter (bone, fish, etc.), dissolve 
residue insol. in NH 4 citrate as in 2.020(c), (d), or (e). 

2.046 Determination 

(a) Gravimetric quinolinium molybdophosphate method 
(11).— -Treat 1 g sample as in 2.045(a) or (b). Transfer aliquot of 
citrate-insol. P 2 5 contg ==25 mg P 2 5 and proceed as in 2.028. 

(b) Spectrophotometric molybdovanadophosphate method 
{15).— Treat 1 g sample as in 2.045(a) or (b). Adjust concn of 
citrate-insol. P 2 5 soln as in 2.024(a) or (b) and proceed as in 
2.025. 

(c) Alkalimetric quinolinium molybdophosphate method 
(16). — Treat 1 g sample by 2.045(a) or (b). Transfer aliquot of 
citrate-insol. P 2 5 contg ^5 mL coned acid to 500 mL erlenmeyer. 
Add 20 mL 10% citric acid soln and dil. to 100 mL with H 2 0. 
Continue as in 2.031(a), beginning "Add 60 mL quimociac 
reagent, . . ." 

2.047 Citrate-Soluble Phosphorus— Official Final Action 

Subtract sum of H 2 0-sol. and citrate-insol. P 2 5 from total P 2 5 
to obtain citrate-sol. P 2 5 . 

Available Phosphorus 

2.048 Indirect Method — Official Final Action 

Subtract citrate-insol. P 2 5 from total P 2 5 to obtain available 
P 2 5 . 

Direct Method { 17)— Official Final Action 

2.049 Reagents 

(Caution: See 51.026, 51.028, 51.030, and 
Acids, Chap. 51.) 

(a) Nitric-perchloric acid mixture.— Add 300 mL 70% HCI0 4 to 
700 mL HN0 3 . 

(b) Ternary acid mixture.— Add 20 mL H 2 S0 4 to 100 mL HN0 3 , 
mix, and add 40 mL 70% HCI0 4 . 

(c) Modified molybdovanadate reagent. — Prep, as in 2.022(a) 
except use 250 mL 70% HCI0 4 instead of 450 mL 



2.050 



Preparation of Solution 



(a) Acidulated samples, mixed fertilizers, and materials con- 
taining water-soluble compounds. — (7) Without filtration of ci- 
trate digest — Remove H 2 0-sol. P 2 5 as in 2.040,collecting filtrate 
in 500 ml vol. flask, but do not add HN0 3 to filtrate. Treat H 2 0- 
insol. residue with NH 4 citrate soln as in 2.045(a). Exactly 1 hr 
after adding filter and residue, remove flask from app. and 
transfer contents to flask contg H 2 0-sol. fraction. Cool to room 
temp, immediately, dil. to vol., mix thoroly, and let stand ^2 hr 
before removing aliquot. 

(2 ) With filtration of citrate digest. — I f d esi red ,washbygravity 
into 500 mL Kohlrausch flask contg 5 mL HN0 3 (1 + 1), catching 
filtrate from insol. residue, 2.045(a), in the Kohlrausch flask 
contg H 2 0-soL fraction, and wash residue until vol. soln in flask 
is ca 500 mL. Cool, dil. to 500 mL, and mix. 



14 



2, Fertilizers 



AOAC Methods (1980) 



(b) Nonacidulated samples. — Place 1 g sample (ground to 
pass No. 40 sieve in case of Ca metaphosphate) on dry 9 cm 
paper. Without previous washing with H 2 0, proceed as in (a)(7) 
or (2). If (2) is used, wash residue until vol. soln is ca 350 ml 
Cool, dil. to 500 ml, and mix. 



2.051 Alkali metric Quinolinium Moiybdophosphate 
Method [16)— Official Final Action 

Treat 1 g sample by appropriate modification of 2.050. Transfer 
aliquot contg =s30 mg P 2 5 and ^10 mL NH 4 citrate soln, 2.044(a), 
to 500 mL erlenmeyer. Dil., if necessary, to 50 mL, add 10 mL 
HN0 3 (1+1), and boil gently 10 min. Cool, dil. to 100 mL, and 
continue as in 2.031(a), beginning "Add 60 mL quimociac 
reagent, . . ." 



2.052 Spectrophotometric Molybdovanadophosphate 

Method (18)— Official Final Action 

(Not applicable to materials yielding colored solns or solns 
contg ions other than orthophosphate which form colored 
complexes with molybdovanadate. Not recommended for basic 

slag.) 

Prep, std curve as in 2.023, using photometer, 2.021. 

Pipet, into 100 mL vol. flasks, 5 mL aliquots std phosphate 
solns contg 2 and 3.5 mg P 2 5 /aliquot, 2.022(b), resp., add 2 mL 
70% HCIO4, and develop color as in 2.023. Adjust instrument to 
zero A for 2 mg std and det. A of 3.5 mg std. {A of latter must 
be practically identical with corresponding value on std curve.) 

Prep, sample as in 2.050. 

(a) Samples containing up to 5% P 2 5 . — Pipet 10 mL sample 
soln into 125 mL erlenmeyer, and treat by one of following 
methods {Caution: See 51.019, 51.026, and 51.028): 

(7) Add 5 mL 20% NaCI0 3 soln and 10 mL HN0 3 -HCIO 4 mixt, 
2.049(a). Boil gently until greenish-yellow color disappears (ca 
20 min), cool, and add 2 mL HCI. After vigorous reaction 
subsides, evap. to fumes of HCI0 4 , and fume 2 min. 

{2) Add 5 mL ternary acid mixt., 2.049(b), swirl, boil gently 15 
min, and digest at 150-200° until clear white salt or colorless 
soln remains. Evap. to white fumes and continue heating 5 min. 

Cool, add 15 mL H 2 0, and boil 5 min. Transfer to 100 mL vol. 
flask, dil. to 50 ml, swirl, and cool to room temp. Add 5 mL std 
phosphate soln contg 2 mg P 2 O s and 20 mL modified molyb- 
dovanadate soln, 2.049(c). Dit. to 100 mL, and continue as in 
2.025(a). 

(b) Samples containing more than 5% P 2 5 . — Dil. soln to such 
vol. that 5-10 mL aliquot contains 2-5 mg P 2 5 . Digest as in 
(a)(7) or [2). Without adding std phosphate soln, continue as in 
(a). 



NITROGEN 
2.054 Detection of Nitrate*— Official Final Action 

Mix 5 g sample with 25 mL hot H 2 0, and filter. To 1 vol. of this 
soln add 2 vols H 2 S0 4 , free from HN0 3 and oxides of N, and let 
cool. Add few drops coned FeSO^soln in such manner that fluids 
do not mix. If nitrates are present, junction at first shows purple, 
afterwards brown, or if only minute amt is present, reddish 
color. To another portion of soln add 1 mL 1% NaNO z soln and 
test as before to det. whether enough H 2 S0 4 was added in first 
test. 



Total Nitrogen 

(Provide adequate ventilation in laboratory and 
do not permit accumulation of exposed Hg.) 



2.055 



Reagents— Official Final Action 



(a) Sulfuric acid.— 93-98% H 2 S0 4 , N-free. 

(b) Mercuric oxide or metallic mercury. — HgO or Hg, reagent 
grade, N-free. 

(c) Potassium sulfate {or anhydrous sodium sulfate). — Re- 
agent grade, N-free. 

(d) Salicylic acid. — Reagent grade, N-free. 

(e) Sulfide or thiosulfate soln.— Dissolve 40 g com. K 2 S in 1 
L H 2 0. (Soln of 40 g Na 2 S or 80 g Na 2 S 2 3 .5H 2 in 1 L may be 
used.) 

(f) Sodium hydroxide. — Pellets or soln, nitrate-free. For soln, 
dissolve ca 450 g solid NaOH in H 2 0, cool, and dil. to 1 L. (Sp gr 
of soln should be ^1.36.) 

(g) Zinc granules. — Reagent grade, 
(h) Zinc dust. — Impalpable powder. 

(i) Methyl red indicat or. —Dissolve 1 g Me red in 200 mL 
alcohol. 

(j) Hydrochloric or sulfuric acid std soln. — 0.5/V, or 0. 1 N when 
amt of N is small. Prep, as in 50.011 or 50.039. 

(k) Sodium hydroxide std soln. — 0.1 N (or other specified 
concn). Prep, as in 50.032-50.034. 

Stdze each std soln with primary std, Chap. 50, and check one 
against the other. Test reagents before use by blank detn with 
2 g sugar, which ensures partial reduction of any nitrates 
present. 

Caution: Use freshly opened H 2 S0 4 or add dry P 2 5 to avoid 
hydrolysis of nitriles and cyanates. Ratio of salt to acid (wt:vol.) 
should be ca 1:1 at end of digestion for proper temp, control. 
Digestion may be incomplete at lower ratio; N may be lost at 
higher ratio. Each g fat consumes 10 mL H 2 S0 4 , and each g 
carbohydrate 4 mL H 2 S0 4 during digestion. 



2.053 Gravimetric Quinolinium Moiybdophosphate 
Method {19)— Official Final Action 

(a) Solns containing no organic phosphorus. — Prep, sample 
as in 2.050. Pipet, into 500 mL erlenmeyer, aliquot contg ^25 
mg P 2 5 and =s10 mL original NH 4 citrate soln. Dil., if necessary, 
to ca 50 mL, add 10 mL HN0 3 (1+1), and boil gently 10 min. 
Cool, dil. to 150 mL, and proceed as in 2.028(a) or (b). 

(b) Solns containing organic phosphorus. — {Caution: See 
51.019, 51.026, and 51.028.) Select aliquot as in (a). Add 10 mL 
20% NaCI0 3 and 10 mL HN0 3 -HCI0 4 mixt., 2.049(a). Boil vigor- 
ously until greenish-yellow color disappears (usually ca 30 min), 
cool, and add 2 mL HCI. After vigorous reaction subsides, evap. 
to white fumes, and continue heating 5 min. Cool, and proceed 
as in 2.028(a) or (b). 



2.056 Apparatus — Official Final Action 

(a) For digestion. — Use Kjeldahl flasks of hard, moderately 
thick, well-annealed glass with total capacity ca 500-800 mL. 
Conduct digestion over heating device adjusted to bring 250 mL 
H 2 at 25° to rolling boil in ca 5 min or other time as specified 
in method. To test heaters, preheat 10 min if gas or 30 min if 
elec. Add 3-4 boiling chips to prevent superheating. 

(b) For distillation.— Use 500-800 mL Kjeldahl or other suit- 
able flask, fitted with rubber stopper thru which passes lower 
end of efficient scrubber bulb or trap to prevent mech. carryover 
of NaOH during distn. Connect upper end of bulb tube to 
condenser tube by rubber tubing. Trap outlet of condenser in 
such way as to ensure complete absorption of NH 3 distd over 
into acid in receiver. 



AOAC Methods (1980) 



Nitrogen 



15 



2.057 Improved Kjeldahl Method for Nitrate-Free 
Samples {20)— Official Final Action 

{Caution: See 51.030 and 51.065.) 

Place weighed sample (0.7-2.2 g) in digestion flask. Add 0.7 
g HgOor0.65g metallic Hg, 15 g powd K 2 S0 4 or anhyd. Na 2 S0 4 , 
and 25 mL H 2 S0 4 . If sample >2.2 g is used, increase H 2 S0 4 by 10 
mL for each g sample. Place flask in inclined position and heat 
gently until frothing ceases (if necessary, add small amt of 
paraffin to reduce frothing); boil briskly until soln clears and 
then ^30 min longer (2 hr for samples contg org. material). 

Cool, add ca 200 mL H 2 0, cool <25°, add 25 mL of the sulfide 
or thiosulfate soln, and mix to ppt Hg. Add few Zn granules to 
prevent bumping, tilt flask, and add layer of NaOH without 
agitation. (For each 10 mL H 2 S0 4 used, or its equiv. in dild H 2 S0 4 , 
add 15 g solid NaOH or enough soln to make contents strongly 
alk.) (Thiosulfate or sulfide soln may be mixed with the NaOH 
soln before addn to flask.) Immediately connect flask to distg 
bulb on condenser, and, with tip of condenser immersed in std 
acid and 5-7 drops indicator in receiver, rotate flask to mix 
contents thoroly; then heat until all NH 3 has distd (^150 mL 
distillate). Remove receiver, wash tip of condenser, and titr. 
excess std acid in distillate with std NaOH soln. Correct for blank 
detn on reagents. 

% N - [(mL std acid x normality acid) - (mL std NaOH 

x normality NaOH)] x1.4007/g sample 

2 .058 Impro ved Kjeldahl Method for Nitrate-Containing 
Samples — Official Final Action 

(Not applicable to liqs or to materials with high 
CI:N0 3 ratio. Caution: See 51.030 and 51.065.) 

Place weighed sample (0.7-2.2 g) in digestion flask. Add 40 
mL H 2 S0 4 contg 2 g salicylic acid. Shake until thoroly mixed and 
let stand, with occasional shaking, ^30 min; then add (7) 5 g 
Na 2 S 2 3 .5H 2 or [2) 2 g Zn dust (as impalpable powder, not 
granulated Zn or filings). Shake and let stand 5 min; then heat 
over low flame until frothing ceases. Turn off heat, add 0.7 g 
HgO (or 0.65 g metallic Hg) and 15 g powd K 2 S0 4 (or anhyd. 
Na 2 S0 4 ), and boil briskly until soln clears, then ^30 min longer 
(2 hr for samples contg org. material). 

Proceed as in second par. of 2.057. 

Comprehensive Nitrogen Method (27) 
Official Final Action 

(Applicable to all fertilizer samples. 
Caution: See 51.030 and 51.079.) 

2.059 Reagents 

(a) Chromium metal. — 100 mesh, low N (Fisher Scientific Co. 
No. C-318 or Sargent-Welch Scientific Co. No. SC11432 is satis- 
factory). 

(b) Alundum. — Boiling stones. 8-14 mesh (Arthur H. Thomas 
Co. No. 1590-D18, or equiv.). 

(c) Dilute sulfuric acid.— Slowly add 625 mL H 2 S0 4 to 300 mL 
H 2 0. Dil. to ca 1 L and mix. After cooling, dil. to 1 L with H 2 and 
mix. Avoid absorption of NH 3 from air during prepn, particularly 
if stream of air is used for mixing. 

(d) Sodium thiosulfate or potassium sulfide soln.— 160 g 
Na 2 S 2 3 .5H 2 0/L or 80 g K 2 S/L 

For other reagents, see 2.055. 



occasional gentle swirling to dissolve all nitrate salts. Add 7 mL 
HCI and let stand ^30 sec but ==10 min. 

Place flask on preheated burner with heat input set at 7.0-7.5 
min boil test, 2.056(a). After heating 3.5 min, remove from heat 
and let cool. 

Add 22 g K 2 S0 4 , 1.0 g HgO, and few granules Alundum. Add 
40 mL dil. H 2 S0 4 , (c). (If adequate ventilation is available, 25 mL 
H 2 S0 4 may be added instead of dil. H 2 S0 4 . If org. matter which 
consumes large amt of acid exceeds 1.0 g, add addnl 1.0 mL 
H 2 S0 4 for each 0.1 g org. matter in excess of 1.0 g.) 

Place flask on burners set at 5 min boil test. (Pre-heated 
burners reduce foaming with most samples. Reduce heat input 
if foam fills ^ 2 / 3 of bulb of flask. Use variable heat input until 
this phase is past.) Heat at 5 min boii test until dense white 
fumes of H 2 S0 4 clear bulb of flask. Digestion is now complete for 
samples contg ammoniacal, nitrate, and urea N. For other 
samples, swirl flask gently and continue digestion 60 min more. 

Proceed as in 2.057, second par., substituting 2.059(d) for 
2.055(e). 

Modified Comprehensive Nitrogen Method (22) 
Official First Action 

(Applicable to all fertilizer samples) 

2.061 Reagents 

See 2.055(a), (c), (f), (i), (j), (k), 2.059(a), (b), and in addn: 
Copper sulfate pentahydrate {or anhydrous copper sulfate). — 
Reagent grade, N-free. 



2.062 



Determination 



2.060 



Determination 



Place 0.2-2.0 g sample contg ^60 mg nitrate N in 500-800 mL 
Kjeldahl flask and add 1.2 g Cr powder. Add 35 mL H 2 or, with 
liqs, amt to make total vol. 35 ml. Let stand 10 min with 



{Caution: See 51.019 and 51.030.) 

Proceed as in 2.060, par. 1 and 2, using 0.2-1.6 g sample. For 
samples contg orgs other than urea or urea-form, use 5=0.5 g 
sample. 

Add 15 g K 2 S0 4 or 12 g anhyd. Na 2 S0 4 , 0.4 g anhyd. CuS0 4 or 
0.6 g CuS0 4 .5H 2 0, and ca 0.8 g Alundum granules. Add 37 mL 
H 2 S0 4 (1 + 1). (If adequate ventilation is available, 20 mL H 2 S0 4 
may be added instead of H 2 S0 4 (1 + 1). If org. matter other than 
urea exceeds 1.0 g, add addnl 1.0 mL H 2 S0 4 for each 0.1 g fat or 
0.2 g other org. matter in excess of 1.0 g.) 

Proceed as in 2.060, par. 4, substituting 75 min for 60 min in 
last sentence. 

Cool flask until it can be handled without gloves, and add ca 
250 mL H 2 0. Swirl to dissolve contents, and cool <25°. Add ca 
0.8 g Alundum granules to minimize bumping, tilt flask, and 
add layer of NaOH without agitation. (For each 10 ml H 2 S0 4 used, 
or its equiv. in H 2 S0 4 (1 + 1 ), add 1 5 g solid NaOH or enough soln 
to make contents strongly alk.) Proceed as in 2.057, par. 2, 
beginning "Immediately connect flask to distg bulb . . ." 

Raney Powder Method (27) 
Official Final Action 

(Applicable to all fertilizer samples except "nitric phosphates" 
contg nonsulfate S. Caution: See 51.030 and 51.079.) 

2.063 Reagents 

(a) Raney catalyst powder No. 2813.-50% Ni, 50% Al (W. R. 
Grace & Co., Davison Chemical Division, 10 E Baltimore St, 
Baltimore, MD 21203). Caution: Raney catalyst powders react 
slowly in H 2 or moist air to form alumina; avoid prolonged 
contact with air or moisture during storage or use. 

(b) Sulfuric acid-potassium sulfate soln. — Slowly add 200 mL 
H 2 S0 4 to 625 mL H 2 and mix. Without cooling, add 106.7 g 



16 



2. Fertilizers 



AOAC Methods (1980) 



K 2 S0 4 and continue stirring until all salt dissolves. Dil. to ca 1 L 
and mix. Cool, dil. to 1 L with H 2 0, and mix. Avoid absorption 
of NH 3 from air during prepn particularly if stream of air is used 
for mixing. 
For other reagents, see 2.055. 



2.064 



Determination 



Place 0.2-2.0 g sample contg =£42 mg nitrate N in 500-800 mL 
Kjeldahl flask (800 mL flask is preferred with samples which 
foam considerably, especially orgs). Add 1.7 g Raney catalyst 
powder, 3 drops tributy I citrate, and 150 mL H 2 S0 4 -K 2 S0 4 soln. If 
org. matter exceeds 0.6 g, add addnl 2.5 mL of this soln for each 
0.1 g org. matter in excess of 0.6 g. 

Swirl to mix sample with acid and place flask on cold burner. 
If burner has been in use, turn off completely 3=10 min before 
placing flask on burner. After flask is on burner, set heat input 
to 5 min boil test. When sample starts boiling, reduce heat to 
pass 10 min boil test. After 10 min, raise flask to vertical position 
and add 0.7 g HgO and 15 g K 2 S0 4 . (Contents of Kel-Pak No. 5 
(Curtin Matheson Scientific, Inc.) without plastic container may 
be used.) Replace flask in inclined position and increase heat to 
4-5 min boil test. (Reduce heat input if foam fills 2=% of bulb of 
flask. Use variable heat input until this phase is past.) Heat at 
4-5 min boil test until dense white fumes of H 2 S0 4 clear bulb of 
flask. Digestion is now complete for samples contg only am- 
moniacal, nitrate, and urea N. For other samples, swirl flask 
gently and continue digestion addnl 30 min. 

Proceed as in 2.057, second par. If 800 mL Kjeldahl flasks have 
been used, add 300 instead of 200 mL H 2 0. 



Ammoniacal Nitrogen 

2.065 Magnesium Oxide Method — Official Final Action 

(Not applicable in presence of urea) 

Place 0.7-3.5 g, according to NH 3 content of sample, in distn 
flask with ca 200 mL H 2 and 5*2 g carbonate-free MgO. Connect 
flask to condenser by Kjeldahl connecting bulb, distil 100 mL 
liq. into measured amt std acid, 2.055(j), and titr. with std NaOH 
soln, 2.055(k), using Me red, 2.055(i). 

2.066 * Formaldehyde Titration Method * 

Official Final Action 

(Applicable to NH 4 N0 3 and (NH 4 ) 2 S0 4 ) 
See 2.058, 11thed. 



Ind. Eng. Chem. 11, 465(1919)) or other suitable scrubbing bulb 
that will prevent passing over of any spray, connect with 
condenser, tip of which always extends beneath surface of std 
acid in receiving flask. Mix contents of distg flask by rotating. 
Heat slowly at first and then at rate to yield 250 mL distillate in 
1 hr. Collect distillate in measured amt std acid, 2.055(j), and 
titr. with std NaOH soln, 2.055(k), using Me red, 2.055(i). 

Nitrate Nitrogen 

2.069 Robertson Method (24)— Official Final Action 

(Applicable in presence of Ca cyanamide 
and urea. Caution: See 51.030 and 51.065.) 

(a) Det. total N as in 2.058 7 2.060, or 2.064. 

(b) Det. H 2 0-insol. N as in 2.072, but use 2.5 g sample. Dil. 
filtrate to 250 mL 

(c) Place 50 mL portion filtrate in 500 mL Kjeldahl flask and 
add 2 g FeS0 4 .7H 2 and 20 mL H 2 S0 4 . (If total N is >5%, use 5 
g FeS0 4 .7H 2 0.) Digest over hot flame until all H 2 is evapd and 
white fumes appear, and continue digestion at least 10 min to 
drive off nitrate N. If severe bumping occurs, add 10-15 glass 
beads. Add 0.65 g Hg, or 0.7 g HgO, and digest until all org. 
matter is oxidized. Cool, dil., add the K 2 S soln, and complete 
detn as in 2.057. Before distn, add pinch of mixt. of Zn dust and 
granular "20-mesh" Zn to each flask to prevent bumping. 

Total N (a) - H 2 0-insol. N (b) = H 2 0-sol. N. 
H 2 0-sol. N - N obtained in (c) - nitrate N. 

2.070 Jones Modification of Robertson Method (24) 
Official Final Action 

(Applicable when H 2 0-sol. N need not be detd. 
Caution: See 51.030 and 51.065.) 

Weigh 0.5 g sample into Kjeldahl flask, add 50 mL H 2 0, and 
rotate gently. Add 2 g FeS0 4 .7H 2 and rotate. Add 20 mL H 2 S0 4 . 
Digest over hot flame. When H 2 evaps and white fumes appear, 
add 0.65 g Hg and proceed as in 2.057. 

Total N - N thus found = nitrate N. 

2.071 * Water-insoluble Nitrogen in Cyanamide {25) & 

Official Final Action 

See 2.063, 11th ed. 

Water-Insoluble Nitrogen 

{See 2.079(a) and (b) for urea-formaldehyde 
or mixts contg such compds.) 



Nitrate and Ammoniacal Nitrogen 

2.067 • Ferrous Su/fate-Zinc-Soda Method * 
Official Final A ction 

(Not applicable in presence of org. matter, 
Ca cyanamide, and urea) 

See 2.059, 11th ed. 



2.068 Devarda Method (23)— Official Final Action 

(Not applicable in presence of org. matter, 
Ca cyanamide, and urea) 

Place 0.35 or 0.5 g sample in 600-700 mL flask and add 300 
mL H 2 0, 3 g Devarda alloy (Cu 50, Al 45, Zn 5), and 5 mL NaOH 
soln (42% by wt), pouring latter down side of flask so that it 
does not mix at once with contents. By means of Davisson (J. 



2.072 Method I— Official Final Action 

Place 1 or 1.4 g sample in 50 mL beaker, wet with alcohol, add 
20 mL H 2 0, and let stand 15 min, stirring occasionally. Transfer 
supernate to 11 cm Whatman No. 2 paper in 60° long-stem 
funnel 60 mm diam., and wash residue 4 or 5 times by decanting 
with H 2 at room temp. (20-25°). Finally transfer all residue to 
filter and complete washing until filtrate measures 250 mL. Det. 
N in residue as in 2.057. 



Method 11 &6)— Official First Action 

2.073 Apparatus 

Extraction tube. — Glass, 250 x 10 mm id, 12 mm od, con- 
stricted to 3-4 mm at one end. 



2.074 



Determination 



it Surplus method—see inside front cover. 



Weigh 3.0 g unground mixed sample and place in extn tube 
contg small glass wool plug. Place addnl glass wool pad on top 



AOAC Methods (1980) 



Nitrogen 



17 



of sample. Connect 250 or 500 mL separator to column with 75 
mm piece of rubber tubing. Close stopcock of separator and 
add 250 mL deionized H 2 0. Open stopcock and let quick rush of 
H 2 pass thru column. After initial rush of H 2 0, close stopcock. 
Adjust flow thru stopcock to ca 2 mL/min. Squeeze rubber 
connection to bring level of H 2 ca 25 mm above column bed. 
System then operates as constant-head feeder. 

After H 2 wash is complete, disconnect column from rubber 
tubing. Invert column over Kjeldahl flask and force contents into 
flask with aid of pressure bulb. Wash traces of sample from 
tube into Kjeldahl flask and wash sample from walls of digestion 
flask with min. H 2 0. Det. N in residue as in 2.059-2.060 or 
2.063-2.064. 

ir Nitrogen Activity & 

2.075 Removal of Water-Soluble Nitrogen 
Official Final Action 

(a) Mixed fertilizers.— See 2.058, 10th ed. 

(b) Raw materials.—See 2.058, 10th ed. 

2.076 Water-Insoluble Organic Nitrogen 
Soluble in Neutral Permanganate 
Official Final Action 

See 2.059, 10th ed. 

2.077 Water-insoluble Organic Nitrogen 
Distilled from Alkaline Permanganate \£7) 
Official Final Action 

See 2.060-2.061, 10th ed. 



Nitrogen Activity index (Al) of Urea- 
Formaldehyde Compounds (25) 
Official Final Action 

(Applicable to urea-formaldehyde compds 
and mixts contg such compds) 



Urea (29) — Official Final Action 



2.080 



2.078 



Reagent 



Phosphate buffer sofn.—pH 7.5. Dissolve 14.3 g KH 2 P0 4 and 
91.0 g K 2 HP0 4 in H 2 and dil. to 1 L Dil. 100 mL of this soln to 
1 L. 



2.079 



Determination 



(a) Crush sample (do not grind) to pass No. 20 sieve. 

(b) Det. cold H 2 0-insol. N {WIN) as in 2.072, keeping temp, at 
25±2°. Stir at 5 min intervals during 15 min standing. 

(c) Det. hot H 2 0-insol. N {HWIN) in phosphate buffer soln as 
follows: Place accurately weighed sample contg 0.1200 g WIN 
in 200 mL tall-form beaker. Add ca 0.5 g CaC0 3 to mixed 
fertilizers contg urea-HCHO compds. From supply of boiling 
buffer soln, add 100 mL from graduate to sample, stir, cover, 
and immerse promptly in boiling H 2 bath so that liq. in beaker 
is below H 2 level in bath. Maintain bath at 98-100°, checked 
with thermometer, and stir at 10 min intervals. After exactly 30 
min, remove beaker from bath and filter promptly thru 15 cm 
Whatman No. 12 fluted paper. If filtration takes >4 min, discard 
detn. Repeat detn, stirring in 1 g Celite filter-aid just before 
removing beaker from bath, and filter. 

Wash insol. residue completely onto paper with boiling H 2 
and continue washing until total vol. used is 100 mL. Complete 
washing before filtrate becomes cloudy or its temp, drops to 
<60°. Det. total N {HWIN) in wet paper and residue as in 2.057, 
using 35 mL H 2 S0 4 when CaC0 3 has been added. 

Activity index {Al) = {%WIN - %HWIN) x 100/%W//V 



Reagent 



Neutral urease soln. — Use fresh com. 1% urease soln, or 
dissolve 1 g urease powder in 100 mL H 2 0, or shake 1 g jack 
bean meal with 100 mL H 2 5 min. Transfer 10 mL soln to 250 
mL erlenmeyer, dil. with 50 mL H 2 0, and add 4 drops Me purple 
(available from Fisher Scientific Co.; No. So-l-9). Titr. with 0.1 N 
HCI to reddish purple; then back-titr. to green with 0.1/V NaOH. 
From difference in mL, calc. vol. 0.1/V HCI required to neutze 
remainder of soln (usually ca 2.5 mL/100 mL), add this amount 
of acid, and shake well. 

Verify enzyme activity of urease source periodically. Discard 
any source which does not produce soln capable of hydrolyzing 
0.1 g urea/20 mL soln. 



2.081 



Determination 



Weigh 10±0.01 g sample and transfer to 15 cm Whatman No. 
12 fluted filter paper. Leach with ca 300 mL H 2 into 500 mL vol. 
flask. Add 75-100 mL satd Ba(OH) 2 soln to ppt phosphates. Let 
settle and test for complete pptn with few drops satd Ba(OH) 2 
soln. Add 20 mL 10% Na 2 C0 3 soln to ppt excess Ba and any sol. 
Ca salts. Let settle and test for complete pptn. Dil. to vol., mix, 
and filter thru 15 cm Whatman No. 12 fluted paper. Transfer 50 
mL aliquot (equiv. to 1 g sample) to 200 or 250 mL erlenmeyer 
and add 1-2 drops of Me purple. Acidify with 2/V HCI and add 
2-3 drops excess. Neutze soln with 0.1 N NaOH to first change 
in color of indicator. Add 20 mL neutral urease soln, close flask 
with rubber stopper, and let stand 1 hr at 20-25°. Cool flask in 
ice-H 2 slurry and titr. at once with 0.1/V HCI to full purple; then 
add ca 5 mL excess. Record total vol. added. Back-titr. excess 
HCI with 0.1/V NaOH to neut end point. 

% Urea = (mL 0.1/V HCI - mL 0.1/V NaOH) x 0.3003. 

Biuret 

Spectrophotometry Method (30) 
Official First Action 

(Applicable to urea only. Do not use for mixed fertilizers) 



2.082 



Reagents 



(a) Alkaline tartrate soln. —Dissolve 40 g NaOH in 500 mL H 2 0, 
cool, add 50 g NaKC 4 H 4 6 .4H 2 0, and dil. to 1 L. Let stand 1 day 
before use. 

(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in C0 2 - 
free H 2 and dil. to 1 L 

(c) Biuret std soln. — 1 mg/mL. Dissolve 100 mg reagent grade 
biuret in C0 2 -free H 2 and dil. to 100 mL. Biuret may be purified 
as follows: Dissolve 10 g in 1 L absolute alcohol and cone, by 
gentle heating to ca 250 mL. Cool at 5° and filter thru gooch 
(60% recovery). Repeat crystn and dry final product in vac. oven 
at 80°. 

2.083 Preparation of Standard Curve 

Transfer series of aliquots, 2-50 mL, of std biuret soln to 100 
mL vol. flasks. Adjust vol. to ca 50 mL with C0 2 -free H 2 0, add 
1 drop Me red, and neutze with 0.1/V H 2 S0 4 to pink color. Add, 
with swirling, 20 mL alk. tartrate soln and then 20 mL CuS0 4 
soln. Dil. to vol., shake 10 sec, and place in H 2 bath 15 min at 
30±5°. Also prep, reagent blank. Det. A of each soln against 
blank at 555 nm (instrument with 500-570 nm filter is also 
satisfactory) with 2—4 cm cell. Plot std curve. 



* Surplus method— see inside front cover. 



18 



2. Fertilizers 



AOAC Methods (1980) 



2.084 



Determination 



Continuously stir =£10 g sample contg 30—125 mg biuret in 
150 mL ca 50° H 2 30 min. Filter and wash into 250 mL vol. 
flask, and dii. to vol. Transfer 50 mL aliquot to 100 mL vol. flask 
and proceed as in 2.083. 

Atomic Absorption Spectrophotometry Method [31) 
Officiai Finai Action 

2.085 Apparatus and Reagents 

(a) Atomic absorption spectrophotometer. — IL Model 353 (In- 
strumentation Laboratory, Inc., 113 Hartwell Ave, Lexington, MA 
02173), or equiv., with Cu hollow cathode lamp. 

(b) Copper sulfate soln. — Dissolve 15 g CuS0 4 .5H 2 in H 2 
and dil. to 1 L. 

(c) Buffer so/a?.— pH 13.4. Dissolve 24.6 g KOH and 30 g KCI 
in H 2 and dil. to 1 L. 

(d) Starch soln. —Treat 1 g sol. starch with 10 mL cold H 2 0, 
triturate to thin paste, and pour gradually into 150 mL boiling 
H 2 contg 1 g oxalic acid. Boil until soln clears, cool, and dil. to 
200 mL. Prep, fresh weekly. 

(e) Bromocresol purple indicator. — Dissolve 0.1 g bromocre- 
sol purple in 19 mL 0.1/V NaOH and dii. to 250 mL with H 2 0. 

(f) Biuret — To recrystallize, weigh ca 10 g reagent grade 
biuret, transfer to 800 mL beaker, add 500 mL H 2 0, and heat on 
hot plate with occasional stirring until dissolved. Boil until vol. 
decreases to ca 250 mL. Remove, and let cool gradually to room 
temp. Filter thru fritted-glass funnel, transfer to evapg dish, and 
dry 1 hr in 105-1 10° oven. Remove from oven, place in desiccator, 
and coot to room temp. 

(g) Biuret std soln. — 0.4 mg/mL. Dissolve 0.4000 g recrystd 
biuret in warm H 2 0, cool, transfer to 1 L flask, and dil. to vol. 

(h) Copper std solns. — Dil. aliquots of Cu stock soln, 2.110(b), 
with H 2 to obtain 5=4 std solns within range of detn, 1-4 ju,g 
Cu/mL final soln. 



2.086 



Determination of Calibration Factor 



Transfer aliquots of biuret std soln contg 4, 8, and 12 mg 
biuret to sep. 100 mL vol. flasks, dil. to ca 30 mL with H 2 0, and 
add 25 mL alcohol to each. While stirring with mag. stirrer, add 
2 mL starch soln, 10 mL CuS0 4 soln, and 20 mL buffer soln. 
Remove stirring bar, rinse, dil. to vol., mix thoroly, and let stand 
10 min. With vac, filter ca 50 mL thru dry 150 mL medium 
porosity fritted glass funnel into dry flask. Transfer 25 mL 
aliquots of each filtrate to 250 mL vol. flasks, acidify with 5 mL 
1/V HCI, and dil. to vol. with H 2 0. Proceed as in 2.109-2.113, 
using std solns, 2.085(h), to det. complexed Cu in soln by AA 
spectrophotometry after adding equiv. amts of alcohol, KOH 
soln, buffer soln, and 1/V HCI. Take ^3 readings of each soln. 
From mean value of Cu concn, calc. factor relating mg Cu found 
to mg biuret added. Redet. daily. 



2.087 



Determination 



(a) In urea. — Accurately weigh sample contg <10 mg biuret, 
dissolve in H 2 0, transfer to 100 mL vol. flask, add 25 mL alcohol, 
and proceed as in 2.086, beginning "While stirring with mag. 
stirrer, . . ." From Cu found, calc. biuret concn, using factor. 

(b) In mixed fertilizers. — Transfer accurately weighed sample 
contg <40 mg biuret to 250 mL beaker and add 1 mL H 2 for 
each g of sample (5 g max.). Warm, add 65 mL alcohol and 7 
drops bromocresol purple, and adjust pH to first blue color (pH 
6-7) with 20% KOH. Place on hot plate, heat to bp, cool, and, 
if pH has changed, make final adjustment to first blue. Vac-filter 
thru alcohol-washed paper pulp pad into 100 mL vol. flask. (If 
filtrate is not clear, improper pH adjustment has been made. 



Add HCI and readjust to pH 6-7.) Wash pad and ppt with alcohol 
and dil. to vol. with alcohol. Transfer 25 mL aliquot to 100 mL 
vol. flask, and proceed as in 2.086, beginning "While stirring 
with mag stirrer, . . ." From Cu found, calc. biuret concn, using 
factor and appropriate diln factors. (Final aliquot can be varied 
to give Cu concn between 1 and 4 fxg/ml.) 

POTASSIUM 

2.088 * Lindo-G/adding Method {32) * 

Officiai Finai Action 

Gravimetric detn as K 2 PtCI 6 . See 2.076-2.078, 11th ed. 

2.089 * Wet-Digestion Method {33) * 

Official Final Action 

Pptn as K 2 PtCI 6 after digestion with HN0 3 and HCI. See 
2.079-2.080, 11th ed. 

2.090 * Recovery of Platinum {34) * 

Procedure 

See 2.081-2.083, 11th ed. 

Flame Photometric Method {35) 
Official Final Action 



{Caution: See 51.007.) 



2.091 



Reagents and Apparatus 



(a) Ammonium oxalate soln. — Dissolve 40 g (NH 4 ) 2 C 2 04 in 1 
L H 2 0. 

(b) Methyl red indicator. — Dissolve 0.2 g Me red in 100 mL 
alcohol. 

( c) Dilute nitric acid. —(1+10). 

(d) Anion exchange resin.— REXYN 203(OH) (Fisher Scientific 
Co.); Duolite A-7 or Duolite A-41 (Diamond Shamrock, 1100 
Superior Ave, Cleveland, OH 44114); Permutit S-100 (Permutit 
Co., E49 Midland Ave, Paramus, NJ 07652); or equiv. 

(e) Potassium nitrate or potassium chloride. — Recrystallize 
reagent grade salt twice from H 2 and dry 5 hr at 105°. 

(ff) Ion exchange column. — Made from 30 cm length of std 
wall glass tubing, 2.5 cm od; one end closed by 1-hole No. 4 
rubber stopper thru which is inserted 2-way stopcock or glass 
tubing connected to rubber tubing and compressor clamp. Do 
not let stopcock tubing protrude above stopper. Choose stopper 
large enough so that there is no space between stopper vertex 
and column wall. Alternatively use glass chromatgc tube 300 x 
19 mm id with stopcock or valve at bottom to control flow rate 
(such as SGA Scientific Inc. No. C-4225). 

Place glass wool plug in bottom of tube, close valve, and add 
H 2 to ht of 10 cm. Transfer portion of resin to 200 mL beaker 
and suspend in H 2 0. Transfer slurry to column and adjust ht of 
packed resin to 20 cm, draining excess H 2 until 2.5 cm head 
remains. Regenerate resin after 10 successive aliquots have 
passed thru. For Na, regenerate after 5 aliquots have passed 
thru. 

2.092 Preparation of Resin 

Place ca 450 g resin in 4 L beaker and add 2 L 5% NaOH. Stir 
30 min with elec. stirrer. Let resin settle, and decant NaOH soln. 
Repeat treatment with 5% NaOH twice, decanting NaOH soln 
after final treatment. Add 2 L H 2 to resin, stir few min, let resin 
settle, and decant wash H 2 0. Repeat 3-4 times. Resin is now in 



it Surplus method— see inside front cover. 



AOAC Methods (1980) 



Potassium 



19 



free base form. Regenerate to nitrate form by treating 3 times 
with HN0 3 (1 + 19), in same manner as with NaOH soln. Wash 
resin with H 2 until pH of washings is s*2 by backwashing in 
column or by stirring and decanting in large beaker. Store resin 
under H 2 in stoppered bottle. 

2.093 Preparation of Solution 

(a) Mixed fertilizers and potassium-magnesium sulfate. — 
Weigh 1.5058 g sample into 250 mL vol. flask (500 mL flask if 
sample contains >30% K 2 0), add 125 mL H 2 and 50 mL 
(NHJ2C2O4 soln, and boil 30 min. Cool, dil. to vol., mix, and pass 
thru dry filter. 

(b) Potassium chloride and sulfate. — Dissolve 1.5058 g in H 2 
and dil. to 500 mL. 

2.094 Preparation of Standard Curve 

Dissolve 1.2931 g KN0 3 (or 0.9535 g KCI) in H 2 and dil. to 500 
mL (1000 ppm K). Prep, std solns by diln to cover range 0-80 
ppm K at intervals =£10 ppm, adding appropriate amt LiN0 3 if 
internal std instrument is to be used. Prep, std curve of emission 
against concn, adjusting instrument so that 50 ppm K gives 
reading near mid-scale. Atomize portions of std solns until 
readings for series are reproducible. 

2 .095 Determination 

(a) Mixed fertilizers, potassium sulfate, and potassium-mag- 
nesium sulfate.— -Transfer 10 mL aliquot of sample soln to 250 
mL beaker. Add 1 drop Me red and neutze with HN0 3 (1+10). 
Adjust H 2 level in column to top of resin and quant, transfer 
aliquot to column. Open stopcock to give flow rate of 2 drops/ 
sec, collecting effluent in 250 mL vol. flask. Wash aliquot into 
resin with 2-3 small portions H 2 0. Collect 50-75 mL effluent; 
then open stopcock and collect addnl 100 mL by pouring H 2 
onto column, making certain that H 2 level does not fall below 
top of resin bed. Dil. to vol. and mix (if internal std instrument 
is used, add required amt LiN0 3 before dilg to vol.). Atomize 
portions of sample several times to obtain reliable av. reading 
for each soln. Det. ppm K from std curve. {Temp, of std and 
sample solns must not differ by >2°.) Calc. % K 2 as follows: 

0-30%: ppm K/2 - %K 2 
>30%: ppm K/1 = %K 2 

(b) Potassium chloride.— Proceed as in (a) but omit neutzn 
and resin treatment. 

2.096 Instrument and Procedure 

Performance Test 

Weigh 1.5058 g K acid phthalate (primary std; %K 2 = 23.0) 
and transfer to 250 mL vol. flask. Add ca 0.5 g <NH 4 ) 2 HP0 4 and 
proceed as in 2.093(a), beginning ". . . add 125 mL H 2 . . ." 



Table 2:03 Aliquots and factors for 
potassium determination 



% K 2 Expected 


Aliquot 


Factor 


2 


no diln 


0.0500 


3-4 


150 


0.08333 


5-6 


100 


0.1250 


7-8 


75 


0.1667 


9-13 


50 


0.2500 


14-16 


40 


0.3125 


17-20 


30 


0.4167 


21-25 


25 


0.5000 


26-30 


20 


0.6250 


31-43 


15 


0.8333 


44-65 


10 


1.250 



Automated Flame Photometric Method {36) 
Official Final Action 

{Caution: See 51.007.) 

2.097 Apparatus 

Automatic analyzer. — Auto Analyzer with following modules 
(available from Technicon Corp.): Sampler II, proportioning 
pump, flame photometer, range expander, and recorder. As- 
semble app. as in Fig. 2:07. 



2,098 



Reagents 



(a) Ammonium oxalate soln. — 4%. See 2.091(a). 

(b) Ammonium citrate soln. — Prep, as in 2.044(a). 

(c) Lithium nitrate soln. —Dissolve 0.6894 g dried (2 hr at 105°) 
LiN0 3 in 1 L H 2 0. 

(d) Potassium std solns. — (7) Stock soln. — 0.5 mg K 2 0/mL. 
Dissolve 1.4447 g dried (2 hr at 105°) KH 2 P0 4 in H 2 and dil. to 
1 L. (2) Working solns— 35, 38, 41, 44, 47, 50, and 55 ^g K 2 0/mL 
Accurately measure 35-55 mL stock soln into 500 mL vol. flasks 
contg 0.2 g {NH 4 ) 2 C 2 4 if samples are prepd by 2.099(a), or 12 
mL NH 4 citrate soln, (b), if by 2.099(b). Dil. to vol. with H 2 and 
mix. (If std solns contg citrate must be held overnight, add 3-4 
drops CHCI3.) 

2.099 Preparation of Samples 

(a) Ammonium oxalate extraction.— Weigh 1 g sample into 
500 mL vol. flask, add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL H 2 0, 
boil 30 min, and cool. Dil. to vol. with H 2 0, mix, and filter or let 
stand until clear. 

(b) Ammonium citrate extraction from direct available phos- 
phorus extract — Prep, as in 2.050. (If solns must be held 
overnight, add 3-4 drops CHCI 3 .) 



2.100 



Calibration 



Pump 55 fxg K 2 0/mL std working soln continuously thru 
system. Set range expander to 1 x position and adjust calibration 
control on flame photometer to read 85% T on recorder. Pump 
35 fig/ml soln continuously and set range expander to 2x 
position. Turn adjustable range positioner or range expander to 
obtain 23% T on recorder. Range of 35-55 /xg K 2 0/mL will read 
ca 20-80% T on recorder with expander set at 2x. (Expansion 
from Model II flame photometers may be <20-80% T.) 



2.101 



Determination 



Pipet aliquot sample soln as indicated in Table 2:03 into 250 
mL vol. flask. Dil. to vol. with H 2 and mix. Place 35-55 /tg/mL 
working std solns in sampler tray before samples. Place 44 
/xg/mL std soln at end of series to check for drift. Read samples 
and stds at rate of 40/hr (2:1 sample-to-wash ratio). Prep, std 
curve of emission against K 2 concn and read tig K 2 0/mL from 
graph. 
% K 2 = {fig K 2 0/mL from graph) x factor from Table 2:03. 



Volumetric Sodium Tetraphenylboron Methods 

Method I {37)— Official Final Action 

2.102 Reagents 

(a) Formaldehyde soln. — 37%. 

(b) Sodium hydroxide so/a?.— 20%. Dissolve 20 g NaOH in 100 
mL H 2 0. 

(c) Sodium tetraphenylboron {STPB) soln. — Approx. 1.2%. 
Dissolve 1 2 g NaB(C 6 H 5 ) 4 in ca 800 mL H 2 0. Add 20-25 g AI(OH) 3 , 



20 



2. Fertilizers 



AOAC Methods (1980) 




RANCH EXPANDER 



FIG. 2:07— Flow schematic for K 2 in fertilizers 

stir 5 min, and filter (Whatman No. 42 paper, or equiv.) into 1 
L vol. flask. Rinse beaker sparingly with H 2 and add to filter. 
Collect entire filtrate, add 2 mL 20% NaOH, dil. to vol. with H 2 0, 
and mix. Let stand 48 hr and stdze. Adjust so that 1 mL STPB 
= 1% K 2 0. Store at room temp. 

(d) Benzalkonium chloride {BAC) soln. — Approx. 0.625%. Dil. 
38 mL 17% Zephiran chloride (Winthrop Laboratories; also 
available at local pharmacies as benzalkonium chloride) to 1 L 
with H 2 0, mix, and stdze. Cetyltrimethylammonium bromide 
may be substituted for Zephiran chloride. If other concn is used, 
adjust vol. 

(e) Clayton Yellow {Titan Yellow; Colour Index No. 19540).— 
0.04%. Dissolve 40 mg in 100 mL H 2 0. 



2.103 



Standardization of Solutions 



(a) BAC soln.— To 1.00 mL STPB soln in 125 mL erlenmeyer, 
add 20-25 mL H 2 0, 1 mL 20% NaOH, 2.5 mL HCHO, 1.5 mL4% 
{NH 4 ) 2 C 2 4 , and 6-8 drops indicator, (e). Titr. to pink end point 
with BAC soln, using 10 mL semimicro buret. Adjust BAC soln 
so that 2.00 mL = 1.00 mL STPB soln. 

(b) Sodium tetraphenylboron soln. — Dissolve 2.500 g KH 2 P0 4 
in H 2 in 250 mL vol. flask, add 50 mL 4% (NH 4 ) 2 C 2 4 soln, dil. 
to vol. with H 2 0, and mix. (It is not necessary to bring to boil.) 
Transfer 15 mL aliquot (51.92 mg K 2 0, 43.10 mg K) to 100 mL 
vol. flask; add 2 mL 20% NaOH, 5 mL HCHO, and 43 mL STPB 
reagent. Dil. to vol. with H 2 0, mix thoroly, let stand 5-10 min, 
and pass thru dry filter. Transfer 50 mL aliquot of filtrate to 125 
mL erlenmeyer, add 6-8 drops indicator, (e), and titr. excess 
reagent with BAC soln. Calc. titer as follows: 

F = 34.61/(43 mL - mL BAC) = % K 2 0/mL STPB reagent. 
Factor F applies to ail fertilizers if 2.5 g sample is dild to 250 mL 
and 15 mL aliquot is taken for analysis. If results are to be 
expressed as K rather than as K 2 0, substitute 28.73 for 34.61 in 
calcg F. 



2.104 



Determination 



Place 2.5 g sample (1.25 g if K 2 >50%) in 250 mL vol. flask, 
add 50 mL 4% (NH 4 ) 2 C 2 4 and 125 mL H 2 0, and boil 30 min. (If 
org. matter is present, add 2 g K-free C before boiling.) Cool, dil. 
to vol. with H 2 0, mix, and pass thru dry filter or let stand until 
clear. Transfer 15 mL aliquot sample soln to 100 mL vol. flask 
and add 2 mL 20% NaOH and 5 mL HCHO. Add 1 mL std STPB 
soln for each 1% K 2 expected in sample plus addnl 8 mL 



excess to ensure complete pptn. Dil. to vol. with H 2 0, mix 
thoroly, let stand 5-10 min, and filter thru dry paper (Whatman 
No. 12 or equiv.). Transfer 50 mL filtrate to 125 mL erlenmeyer, 
add 6-8 drops indicator, (e), and titr. excess reagent with std 
BAC soln. 

% K 2 in sample = (mL STPB added - mL BAC) x F, 
where F = % K 2 0/mL STPB reagent. (Multiply by 2 if 1.25 g 
sample was used.) 

Method II (35)— Official Final Action 

(For use with sample prepd for available P detn) 
2.105 Reagents 

See 2.102(a), (b), (c), (d), and (e). 



2.106 



Standardization of Solutions 



(a) Benzalkonium chloride. — In 125 mL erlenmeyer, add 2.5 
mL neut. NH 4 citrate soln, 2.044(a), 15-20 mL H 2 0, 4 mL HCHO, 
and 2.5 mL 20% NaOH soln. Swirl; then add 4.00 mL STPB soln 
and 6-8 drops indicator, 2.102(e). Titr. to pink end point with 
BAC soln, using 10 mL semimicro buret. Adjust BAC soln so 
that 2.00 mL = 1.00 mL STPB soln. 

(b) Sodium tetraphenylboron soln. — Dissolve 1.4447 g pri- 
mary std KH 2 P0 4 in H 2 in 500 mL vol. flask, add 100 mL neut. 
NH 4 citrate soln, 2.044(a), dil. to vol. with H 2 0, and mix. Transfer 
25 mL aliquot (25.00 mg K 2 0, 20.75 mg K) to 100 mL vol. flask; 
add 8 mL HCHO and 5 mL 20% NaOH, swirl, and add 25 mL 
STPB reagent. Dil. to vol. with H 2 0, mix thoroly, let stand 5-10 
min, and pass thru dry filter. Transfer 50 mL aliquot of filtrate 
to 125 mL erlenmeyer, add 6-8 drops indicator, 2.102(e), and 
titr. excess reagent with BAC soln. Calc. titer as follows: 

F = 25 mg K 2 0/(25 mL STPB - mL BAC) 
= mg K 2 0/mL STPB reagent. 
If results are to be expressed as K rather than K 2 0, substitute 
20.75 for 25 in calcg F. 



2.107 

Prep, as in 2.050. 

2.108 



Preparation of Sample 



Determination 

Transfer 25 mL aliquot of sample soln to 100 mL vol. flask. 
(If org. matter is present, treat 100 mL portion with 1 g K-free 
C and filter before transferring aliquot.) Add 8 mL HCHO first 
and then 5 mL 20% NaOH soln, and wash down sides of flask 
with H 2 0. Swirl and add 1 mL STPB for each 1.5 mg K 2 expected 
in sample aliquot plus addnl 8 mL excess to ensure complete 
pptn. Dil. to vol. with H 2 0, mix thoroly, let stand 5-10 min, and 
pass thru dry filter (Whatman No. 12, or equiv.). Transfer 50 mL 
aliquot filtrate to 125 mL erlenmeyer, add 6-8 drops indicator, 
2.102(e), and titr. excess reagent with std BAC soln. 

% K 2 in sample = (mL STPB added - mL BAC) x F x 2. 

Calcn applies to all fertilizers if 1 g sample is dild to 500 mL 
and 25 mL aliquot is taken fqr analysis. 



OTHER ELEMENTS 

Minor Nutrients by Atomic Absorption 
Spectrophotometry [39)— Official Final Action 

{Caution: See 51.006.) 

2.109 Apparatus and Reagent 

(a) Atomic absorption spectrophotometer. — Several com. 
models are available. Since each design is somewhat different, 



AOAC Methods (1980) 



Other Elements 



21 



with varying requirements of light source, burner flow rate, and 
detector sensitivity, only general outline of operating parameters 
is given in Table 2:04. Operator must become familiar with 
settings and procedures adapted to his own app. and use table 
only as guide to concn ranges and flame conditions. 

(b) Disodium EDTA so//?. —2.5%. Dissolve 25 g Na 2 H 2 EDTA in 
1 L H 2 and adjust to pH 7.0 with 5/V NaOH, using pH meter. 





Table 2:04 Operating 


Parameters 




Wave- 




Range 




Element 


length, A 


Flame 


jiig/mL 


Remarks 


Ca 


4227 


Rich Air-C 2 H 2 


2-20 


1% La, 1% HCI 




4227 


Rich N 2 0-C 2 H 2 


2-20 


Requires special 
burner 


Cu 


3247 


Air-C 2 H 2 


2-20 




Fe 


2483 


Rich Air-C 2 H 2 


2-20 




Mg 


2852 


Rich Air-C 2 H 2 


0.2-2 


May need La 


Mn 


2795 


Air-C 2 H 2 


2-20 




Zn 


2138 


Air-C 2 H 2 


0.5-5 





2.110 



Standard Solutions 



(Do not use <2 mL pipets or <25 mL vol. flasks. Automatic diln 
app. may be used. Prep, std solns in 0-20 /xg range fresh daily.) 

(a) Calcium solns. — (7) Stock so//?.— 25 fxg Ca/mL. Dissolve 
1.249 g CaC0 3 in min. amt 3/V HCI. Dil. to 1 L. Dil. 50 mL to 1 L. 
{2) Working std solns.— 0, 5 ( 10, 15, and 20 ^g Ca/mL contg 1% 
La. To 25 mL vol. flasks add 0, 5, 10, 15, and 20 mL Ca stock 
soln. Add 5 mL La stock soln and dil. to 25 mL. 

(b) Copper stock soln. — 1000/ug Cu/mL. Dissolve 1.000 g pure 
Cu metal in min. amt HN0 3 and add 5 mL HCI. Evap. almost to 
dryness and dil. to 1 L with 0.1/V HCI. 

(c) Iron stock soln. — 1000/ig Fe/mL. Dissolve 1.000 g pure Fe 
wire in ca 30 mL 6/V HCI with boiling. Dil. to 1 L 

(d) Lanthanum stock soln. — 50 g La/L. Dissolve 58.65 g La 2 3 
(99.99%, Ventron Corp., Alfa Products, 8 Congress St, Beverly, 
MA 01915, or equiv.) in 250 mL HCI, adding acid slowly. Dil. to 
1 L. 

(e) Magnesium stock soln. — 1000 fig Mg/mL. Place 1.000 g 
pure Mg metal in 50 mL H 2 and slowly add 10 mL HCI. Dil. to 
1 L. 

(ff) Manganese stock soln. — 1000 ju,g Mn/mL Dissolve 1.582 
g Mn0 2 in ca 30 mL 6/V HCI. Boil to remove CI and dil. to 1 L. 

(g) Zinc stock soln. — 1000 jug Zn/mL. Dissolve 1.000 g pure 
Zn metal in ca 10 mL 6/V HCI. Dil. to 1 L. 

(h) Other std solns. — Dil. aliquots of solns (b), (c), (e), (f), and 
(g) with 0.5/V HCI to make s=4 std solns of each element within 
range of detn. 

2.111 Preparation of Sample Solutions 

{Caution: See 51.019, 51.025, and 51.028.) 

(a) Inorganic materials and mixed fertilizers. — Dissolve 1.00 
g well ground sample in 10 mL HCI in 150 mL beaker. Boil and 
evap. soln nearly to dryness on hot plate. Do not bake residue. 
Redissolve residue in 20 mL 2/V HCI, boiling gently if necessary. 
Filter thru fast paper into 100 mL vol. flask, washing paper and 
residue thoroly with H 2 0. Measure absorption of soln directly, 
or dil. with 0.5/V HCI to obtain solns within ranges of instrument. 
If Ca is to be detd, add enough La stock soln to make final diln 
1% La (i.e., 5 mL La to 25 mL flask, 20 mL to 100 mL flask, etc.). 

(b) Fertilizers containing organic matter (tankage, corncobs, 
cottonseed hulls, etc.). — Place 1.00 g sample in 150 mL beaker 
(Pyrex, or equiv.). Char on hot plate and ignite 1 hr at 500° with 
muffle door propped open to allow free access of air. Break up 
cake with stirring rod and dissolve in 10 mL HCI as in (a). 

(c) Fertilizers containing fritted trace elements.— Dissolve 



*£1.00 g well ground sample in 5 mL HCI0 4 and 5 mL HF. Boil 
and evap. to dense HCI0 4 fumes. Dil. carefully with H 2 0, filter, 
and proceed as in (a). Alternatively, dissolve sample in 10 mL 
HCI, 5 mL HF, and 10 mL MeOH. Evap. to dryness. Add 5 mL HCI 
and evap. Repeat HCI addn and evapn. Dissolve residue as in 
(a). (Normally Pt ware should be used; Pyrex or other glassware 
may be used if Na, K, Ca, and Fe are not to be detd.) 

(d) For manganese. — (7) Acid-soluble, for both Mn +2 and 
Mn+\—See (a), (b), and (c), and 2.139(b). (2) Acid-soluble, for 
Mn +Z only.-^See 2.139(a), 2.140, and 2.141*. (3) Water-soluble, 
for Mn +i only.— See 2.142. 

(e) For iron and zinc— {7) Aqueous extraction.— Place 1.00 g 
sample in 250 mL beaker, add 75 mL H 2 0, and boil 30 min. Filter 
into 100 mL vol. flask, washing paper with H 2 0. Dil. to vol. and 
redil. if necessary. {2) Chelation extraction. — Place 1 .00 g sample 
in 250 mL beaker, and add 5 cm (2") mag. stirrer bar and 100 mL 
2.5% EDTA soln. Stir exactly 5 min, and filter thru Whatman No. 
41 paper, or equiv. If filtrate is cloudy, refilter immediately thru 
fine paper (Whatman No. 5, or equiv.). Redil., if necessary, with 
0.5/V HCI. 



2.112 



Determination 



(P interferes in Ca and may interfere in Mg detn with air-C 2 H 2 

burners. Eliminate interference by adding La stock soln to std 

and sample solns so that final dilns contain 1% La. P does not 

interfere with Ca detn when N 2 0-C 2 H 2 flame is used.) 

Set up instrument as in Table 2:04, or previously established 
optimum settings for app. to be used. Less sensitive secondary 
lines (Gatehouse and Willis, Spectrochim. Acta 17, 710(1961)) 
may be used to reduce necessary diln, if desired. Read ^=4 std 
solns within anal, range before and after each group of 6-12 
samples. Flush burner with H 2 between samples, and re- 
establish absorption point each time. Prep, calibration curve 
from av. of each std before and after sample group. Read concn 
of samples from plot of absorption against /Ltg/mL. 

2.113 Calculations 

% Element = (jug/mL) x (F /sample wt) x 10 -4 , 

F = mL original diln x mL final diln/mL aliquot, if 
original 100 mL vol. is dild. 



Acid-Soluble Boron (40) — Official Final Action 

2.114 Apparatus 

Use high sensitivity glass electrode pH meter for titrn. Use 
assembly with burets, electrodes, and mech. stirrer, arranged 
for convenient use with 250 mL beaker. Use ordinary 50 mL 
burets for the 0.025/V NaOH and 0.02/V HCI. 



2.115 



Reagents 



(a) Boric acid std soln. — Dissolve 1 g H 3 B0 3 in H 2 and dil. to 
1 L 1 mL = 0.1748 mg B. 

(b) Sodium hydroxide std soln. — C0 2 -free, ca 0.025/V. Stdze as 
follows: Pipet 25 mL std H 3 B0 3 soln into 250 mL beaker, add 3.0 
g NaCI, acidify to Me red, dil. to 150 mL, boil to expel C0 2 , cool, 
and titr. potentiometrically as in 2.116. Det. blank by repeating 
titrn, substituting 25 mL H 2 for H 3 B0 3 soln. Calc. B equivalence 
as follows: 

mg B/mL = 4.369/[(mL NaOH soln) - (mL blank)]. 
Protect from atm. C0 2 by soda-lime tubes or other suitable 
means. 

(c) Methyl red indicator. — Dissolve 0.1 g Me red in 50 mL 
alcohol, dil. to 100 mL with H 2 0, and filter if necessary. 



22 



2. Fertilizers 



AOAC Methods (1980) 



2.116 



Determination 



Weigh sample within 1 mg (1.0 g for up to 0.45% B, smaller 
samples for above that content) and place in 250 mL beaker. 
Add ca 50 mL H 2 and 3 mL HCI. Heat to bp and keep hot until 
carbonates are decomposed. Keep soln hot but do not boil 
during following phosphate removal: 

Add 10%Pb[NO z ) 2 $oln, usually 10 mL, or 1 mL for each 1.2% 
P 2 5 if P 2 5 content is known to be >12%. Add NaHC0 3 , little 
at time, until soln approaches neutrality (often observed by 
formation of white ppt in addn to insol. matter already present). 
Add few drops Me red and continue adding NaHC0 3 gradually 
until just alk. to Me red (yellow or very slightly orange). Keep 
mixt. hot but not boiling (H 2 bath or steam bath is best) 30 
min, adding addnl small amts of NaHC0 3 if needed to keep same 
indicator color. (If indicator is bleached by nitrate present, add 
more; if color is obscured by org. matter, use external spot tests 
to follow neutzn.) After neutzn and heating, 40-50 mL soln 
should remain. 

Filter hot soln into 250 mL beaker and wash solids thoroly 
with hot H 2 0, Acidify filtrate with few drops HCI and boil briefly 
to expel most of C0 2 . Neutze hot soln with 0.5A/ NaOH, and 
reacidify with 0.5/V HCI, using 0.3-0.5 mL excess. Dii. to ca 150 
mL and boil gently few min to expel remaining C0 2 . Cool to 
room temp, in running H 2 0. Roughly neutze mixt., using C0 2 - 
free 0.5/V NaOH, and place beaker in titrn assembly with elec- 
trodes and stirrer immersed. Start stirrer and adjust pH to exactly 
6.30 by adding 0.025/V NaOH or 0.02/V HCI as required. (When 
properly adjusted, pH should be steady; drifting usually is due 
to incomplete removal of C0 2 .) When reading of pH 6.30 is 
steady, read 0.025/V NaOH buret, add 20 g mannitol or cryst. r> 
sorbitol, and titr. with 0.025/V NaOH to pH 6.30. (Conveniently 
done with slidewire type instrument by opening pH meter circuit 
when mannitol is added, leaving scale setting at 6.30, closing 
circuit again when indicator color shows that end point is being 
approached, and carefully adding std NaOH soln until galva- 
nometer needle returns to zero. With practice, somewhat slow 
approach to equilibrium, characteristic of glass electrode, can 
be anticipated so as not to overrun end point.) When end point 
is reached, read buret again. Obtain reagent blank by repeating 
detn with all reagents but without sample. 
% B = (mL NaOH soln in detn - mL blank) 

x (mg B/mL NaOH soln)/(10 x g sample) 

2.117 Water-Soluble Boron {40)— Official Final Action 

(Not applicable in presence of >5% urea 
or urea-formaldehyde resins) 

Weigh 2.5 g sample into 250 mL beaker. Add 125 mL H 2 0, boil 
gently ca 10 min, and filter hot thru Whatman No. 40 paper, or 
equiv., into 400 mL beaker. Wash solids well with 6 portions hot 
H 2 and dil. to 5*200 mL with H 2 0. Heat filtrate just to bp. Add 
15 mL 10% BaCI 2 soln to ppt sulfates and phosphates, and add 
powd Ba(OH) 2 , cautiously with stirring, until just alk. to phthln, 
avoiding large excess. Boil in open beaker ^60 min to expel 
NH 3 . (Samples colored by org. matter should be boiled longer.) 
If necessary, add H 2 to keep vol. to ^150 mL. Add and stir 1-2 
teaspoonfuls Filter-Cel or other inert filtering aid, and filter with 
suction thru packed paper pads into 500 mL Pyrex erlenmeyer. 
Wash ppt 6 times with hot boiled H 2 0. (Avoid too large wash 
vols which increase vol. in flask to point of dangerous bumping 
in next step.) 

Make filtrate just colorless to phthln with HCI (1+5), add Me 
red, and make just pink with the acid. Add 5 or 6 boiling stones 
and stirring rod, cover with watch glass, and boil 5 min to 
remove C0 2 . Cool in cold H 2 while covered. Wash cover glass, 
stirrer, and sides of flask. Titr. to yellow of Me red with std 



0.05N NaOH, 50.032-50.036. Add 20 g D-mann/tof and 1 mL or 
more phthln, shake, and wash down sides of flask. Titr. to pink 
end point. Det. blank in exactly same manner as sample. 

1 mL 0.05/V NaOH - 0.000540 g B or 0.00477 g Na 2 B 4 O 7 .10H 2 O. 
Or, (Titer - blank) x factor = lb Na 2 B 4 O 7 .10H 2 O/ton (factor = 
3.807 for 0.05/V NaOH). 

2.118 * Carbonate Carbon {41) — Official Final Action * 

Proceed as in 1.010-1.011, using 2 g sample. Report % C0 2 
by wt. 

Water-Soluble Chlorine {42) 
Official Final Action 

2.119 Reagents 

(a) Silver nitrate std soln. — Dissolve ca 5 g recrystd AgN0 3 in 
H 2 and dil. to 1 L. Stdze against pure, dry NaCI and adjust so 
that 1 mL soln = 0.001 g CI. 

(b) Potassium chromate indicator.— See 50.028(b). 



2.120 



Determination 



Place 2.5 g sample on 11 cm filter paper and wash with 
successive portions boiling H 2 until washings total nearly 250 
mL, collecting filtrate in 250 mL vol. flask. Cool, dil. to vol. with 
H 2 0, and mix well. Pipet 50 mL into 150 mL beaker, add 1 mL 
K 2 Cr0 4 indicator, and titr. with std AgN0 3 soln to permanent red 
of Ag 2 Cr0 4 . 

Acid-Soluble Calcium {43)— Official Final Action 

2.121 Method I 

Weigh 2.5 g sample into 250 mL vol. flask, add 30 mL HN0 3 
and 10 mL HCI, and boil 30 min. Cool, dii. to vol., mix, and filter 
if necessary. Transfer 25 mL aliquot to beaker and dil. to 100 
mL Add 2 drops bromophenol blue, 6.019(f). Add NH 4 OH (1 +4) 
until indicator changes from yellow to green (not blue). If 
overrun, bring back with HCI (1+4). (This gives pH of 3.5-4.0.) 
Dil. to 150 mL, bring to bp, and add 30 mL satd hot (NH 4 ) 2 C 2 4 
soln slowly, stirring constantly. If color changes from green to 
blue or yellow again, adjust to green with HCI (1+4). If yellow, 
adjust with NH 4 OH to green. Digest on steam bath 1 hr, or let 
stand overnight, and cool to room temp. Filter supernate thru 
quant, paper, gooch, or fritted glass filter, and wash ppt thoroly 
with NH 4 OH (1+50). Place paper or crucible with ppt in original 
beaker and add mixt. of 125 mL H 2 and 5 mL H 2 S0 4 . Heat to 
5=70° and titr. with 0.1/V KMn0 4 until first slight pink appears. 
Correct for blank and calc. to Ca. 

2 . 1 22 Method II {A tomic Absorption Method) 
See 2.109-2.113. 

2.123 * Method ill • 

Place CaC 2 4 and filter paper from 2.136 in beaker in which 
pptn was made and dissolve and titr. as in 2.121. 

Cobalt {44)— Official Final Action 
{Caution: See 51.026, 51.028, and Acids, Chap. 51.) 

2.124 Reagents 

(Use H 2 free of interfering elements. Check by shaking 2 drops 

0.01% dithizone in CCI 4 with 10 mL H 2 0. CCI 4 phase should 

remain green.) 

(a) Ternary acid mixture. — See 2.049(b). 
* Surplus method— see inside front cover. 



AOAC Methods (1980) 



Other Elements 



23 



(b) Ammonium hydroxide. — Use fresh stock. (Reagent be- 
comes contaminated with heavy metals on prolonged storage 
in glass.) 

(c) Isoa my I acetate. — Distd. 

(d) 2-Nitroso-1-naphthoi so/a— 0.05%. Dissolve 0.05 g 2-ni- 
troso-1-naphthol in 8 drops 1/V NaOH and 1 mL H 2 0. Add 50-60 
mL H 2 and 6.5-7 mL NH 4 OH, and dil. to 100 mL with H 2 0. 
Divide into 2 ca equal parts and wash each part twice in 100 mL 
centrf. tube with 20 mL isoamyl acetate. Shake 30 sec and centrf. 
after each addn. (It may be necessary to remove part of aq. 
phase to ensure complete removal of foreign matter at interface.) 

(e) Cobaftstdsofns.— (1) Stock soln.— 200 txg Co/mL. Dissolve 
0.0808 g CoCI 2 .6H 2 in H 2 and dil. to 100 mL (2) Working 
soln. — 2 fxg Co/mL. Dil. 1 mL stock soln to 100 mL with H 2 0. 



2.125 



Determination 



Slowly add 20 mL ternary acid mixt. to 2.00 g pulverized, mixed 
fertilizer in 150 mL beaker. Cover with watch glass and digest 
on steam bath overnight. Transfer to hot plate and heat covered 
until dense white fumes appear. (At this point HN0 3 will have 
been expelled. Take care not to lose significant amts of HCI0 4 .) 
Dil. sample contg undissolved residue with H 2 0, transfer to 50 
mL vol. flask, and dil. to vol. Transfer to 100 mL centrf. tube and 
centrf. 5 min at 2000 rpm. Transfer aliquot, contg 2-5 fxg Co, to 
50 mL g-s centrf. tube. Add 10 mL 20% diammonium citrate 
soin and 2 drops phthln. Adjust pH carefully to distinct pink 
with NH 4 0H (1 + 1} and add successively 1 mL 10%Na 2 S 2 O z $oln, 
2 mL 2-nitroso-1-naphthol soln, and 5 mL isoamyl acetate. (Only 
isoamyl acetate addn requires high degree of precision.) Shake 
mixt. 5 min and let sep. Centrf., if necessary. Draw off and 
discard aq. phase thru glass capillary tube attached to vac. 
Wash isoamyl acetate phase with two 5 mL portions 1/V NaOH 
and one 5 mL portion 1/V HCI. Shake 5 min after each addn, let 
layers sep., and draw off and discard aq. phase. Centrf. 2 min 
at 1500 rpm and measure A or %T at 530 nm against isoamyl 
acetate. Det. Co from calibration curve relating A or log %T to 
Co content of std solns contg 0, 2, 4, and 5 /xg Co. 

Iron (45) — Official Final Action 

(Note: Diphenylamine may be harmful. 
Caution: See 51.079 and 51.084.) 

2.126 Reagents 

(a) Diphenylamine soln.— Dissolve 1 g in 100 mL H 2 S0 4 . 

(b) Diphenylamine sulfonate soln. — Dissolve 0.5 g in H 2 in 
100 mL vol. flask and dil. to vol. 

(c) Potassium dichromate std solns. — 0.1/V and 0.0 1/V. Prep. 
0.1/V K 2 Cr 2 7 as in 50.024. Prep. 0.01/V soln by dilg 100 mL 0.1/V 
soln to 1 L. 

(d) Mercuric chloride saturated soin. — Shake HgCI 2 with H 2 
and let settle. 

(e) Stannous chloride soln. — Dissolve 20 g SnCI 2 .2H 2 in 20 
mL HCI, warming gently. Add 20 mL H 2 and dil. to 100 mL with 
HCI (1+1). Keep warm until clear; then add few granules Sn. 
Dispense from dropping bottle. 

2.127 Preparation of Sample Solution 

(a) Suitable for all fertilizers. — Treat 1 g as in 2.020(e), using 
15 mLHCI0 4 . Hold ^1 hratca 170° to remove HN0 3 completely. 
Dil. to 200 mL. 

(b) Suitable for soluble salts and oxides. — Dissolve 1 g in 10 
mL HCI, warming gently. Dil. to 200 mL. 

2.128 Reduction 

Heat aliquot of sample soln (100 mL and 50 mL, resp., for 



samples contg <0.5 and 0.5-4.0% Fe) to bp. Add few drops 
diphenylamine sulfonate soln; then SnCI 2 soln dropwise until 
violet color is discharged and 2 drops excess. (Usually 1-6 drops 
are required. Larger amt may be used with samples contg large 
amt of Fe.) If reduction does not occur, discard and proceed as 
follows with second aliquot: 

Add few granules Zn, boil few min, and either filter off excess 
Zn, washing with hot H 2 0, or let Zn dissolve. Heat to bp and 
finish reduction with SnCI 2 anddiphenylaminesuifonate indicator 
as before. Add 10 mL HCI (1 + 1). Adjust vol. to 75-110 mL with 
H 2 0. Cool rapidly in cold H 2 0. Add 10 mL satd HgCI 2 soln, swirl 
gently, add 5 mL H 3 P0 4 , and titr. immediately. (Small amt of HgCI 
must ppt to ensure complete reduction.) 



2.129 



Titration 



Add 1 drop diphenylamine indicator by pipet (no more; excess 
will interfere with end point if amt of Fe is small). Titr, with 
0.01/V K 2 Cr 2 7 soln. Since end point may be difficult to see with 
very small amt Fe, approach end point slowly, allowing few sec 
for color to develop. Titr. to permanent blue (sometimes green 
with very small amt Fe). For samples contg >4% Fe, use 0.1/V 
K 2 Cr 2 7 fortitrn. 1 mL 0.1/V K 2 Cr 2 7 = 0.00558 g Fe; 1 mL 0.01/V 
= 0.000558 g Fe. 

2. 1 30 A tomic Absorption Method 
See 2.109-2.113. 

Acid-Soluble Magnesium — Official Final Action 

2.131 A tomic Absorption Method 
See2.109i2.113. 

EOT A Titration Method {46) 

(Applicable to samples contg ^0.25% Mn or Zn) 

2.132 Reagents 

Use reagents 1.022(a), (b), (c), (d), (f) (1mL = 1 mg Ca, equiv. 
to 0.6064 mg Mg), (g), (h) (stdzd as in 2.133), and in addn: 

(a) Triethanolamine soln. — ( 1 + 1 ) . 

(b) Potassium ferrocyanide soln.— Dissolve 4 g K 4 Fe(CN) 6 
.3H 2 in 100 mL H 2 0. 

(c) Ferric ammonium sulfate soln. — Dissolve 136 g 
FeNH 4 (S04) 2 .12H 2 in H 2 contg 5 mL H 2 S0 4 , and dil. to 1 L. 
Filter if not clear. 



2.133 



Standardization 



Pipet 10 mL Ca std soln into 300 mL erlenmeyer. Add 100 mL 
H 2 0, 10 mL KOH-KCN soln, 2 drops triethanolamine soln, 5 
drops K 4 Fe(CN) 6 soln, and 15±1 mg calcein indicator. Immedi- 
ately place flask on mag. or other mech. stirrer in front of 
daylight fluorescent light and white background. While stirring, 
titr. with EDTA soln to disappearance of all fluorescent green 
and until soln remains pink. Titr. 3=3 aliquots. From av., calc. Ca 
titer in mg/mL EDTA soln. Ca titer x 0.6064 = Mg titer in mg/mL. 

2.134 Preparation of Solution 

{Caution: See 51.019 and 51.028.) 

(a) Organic materials. — Weigh 1 g sample into 250 mL boiling 
flask or erlenmeyer. Add 5 mL HCI and 10 mL HN0 3 , and boil 
on hot plate or over low flame until easily oxidized org. matter 
is destroyed (ca 15 min). Cool, add 5 mL 70-72% HCI0 4 , and 
heat to appearance of copious fumes and momentary cessation 
of boiling, but not to dryness. Cool, and transfer to 250 mL 



24 



2. Fertilizers 



AOAC Methods (1980) 



beaker with ca 100 ml_ H 2 0. Continue with pH adjustment, as in 
2.135. 

(b) Inorganic materials and mixed fertilizers. — Weigh 1 g 
sample into 250 mL beaker. Add 5 mL HCI and 10 mL HN0 3 . 
Cover with watch glass and heat on asbestos mat on hot plate 
nearly to dryness (ca 30 min). if soln remains colored from org. 
residues, cool, add 5 mL HCI0 4 (70-72%), and continue heating 
to copious fumes and momentary cessation of boiling, but not 
to dryness. 



2.135 



Determination 



Cool prepd soln to room temp. Wash watch glass and inside 
of beaker to ca 100 mL with H 2 0. Using pH meter with glass 
electrode and mech. stirring, adjust to ca pH 3 with 30% KOH 
soln and finally to pH 4.0 with 10% KOH soln. Add FeNH 4 (S0 4 ) 2 
soln, 5 mL for sample <7% P 2 5 , 10 mL for sample 7-15% P 2 5 , 
15 mL for sample 16-30% P 2 5 , and proportionate amts for 
samples >30% P 2 5 . Adjust to pH 5.0 with KOH solns as above, 
or with HCI (1 +4) if pH is >5.0. Cool to room temp, and transfer 
to 250 mL vol. flask with H 2 0. Dil. to vol. with H 2 and mix. Let 
stand until ppt settles. Disturbing ppt as little as possible, filter 
enough soln for aliquots required for titrn thru dry 1 1 cm fluted 
paper, Whatman No. 1, or equiv. 

Pipet two equal aliquots contg <15 mg Ca + Mg (usually 25 
mL) into two 300 mL erlenmeyers and dil. each to 100 mL with 
H 2 0. To one (titrn 7 for Ca + Mg) add 5 mL pH 10 buffer soln, 
2 mL KCN soln, 2 drops triethanolamine soln, 5 drops K 4 Fe(CN) 6 
soln, and 8 drops eriochrome black T indicator. Titr. immediately 
with EDTA soln, stirring and lighting as in stdzn. Color changes 
are wine red, purple, dark blue, to clear pure blue end point, 
becoming green if overtitrd. 

To second aliquot (titrn 2 for Ca) add 10 mL KOH-KCN soln, 
2 drops triethanolamine soln, 5 drops K 4 Fe(CN) 6 soln, and 15±1 
mg calcein indicator. Titr. immediately with EDTA soln as in 
stdzn. 

(Titrn 1 ~ Titrn 2) x Mg titer EDTA x 100/mg sample in 
aliquot = % Mg. 

Titrn 2 x Ca titer EDTA x 10/mg sample in aliquot = % Ca. 

2.136 • Gravimetric Method (47) * 

Official Finai Action 

Removal of Ca as oxalate, pptn as MgNH 4 P0 4 , and ignition to 
Mg 2 P 2 7 . See 2.123, 11th ed. 

2.137 * Volumetric Method— Official * 

Finai Action 

Titrn of pptd MgNH 4 P0 4 with acid. See 2.124, 11th ed. 

2.138 Water-Soluble Magnesium (47) 
Official Final Action 

(a) In potassium-magnesium sulfate, magnesium sulfate, and 
kieserite. — Weigh 1 g sample into 250 mL vol. flask, add 200 mL 
H 2 0, and boil 30 min. Cool, dil. to vol. with H 2 0, and mix. If detn 
is to be conducted gravimetrically, 2.136*, or volumetrically, 
2.137*, see 2.125, 11th ed. 

(b) In other materials, including mixed fertilizers. — Weigh 1 
g sample into 500 mL vol. flask, add 350 mL H 2 0, and boil 1 hr. 
Cool, dil to vol., mix, and filter if necessary. If detn is to be 
conducted gravimetrically, 2.136*, or volumetrically, 2.137*, 
see 2.125, 11th ed. 

(c) By EDTA method. — Transfer aliquot soln prepd as in (a) 
or (b) to beaker and det. Mg as in 2.135, using HCI or KOH to 
adjust pH. 

* Surplus method— see inside front cover. 



Acid-Soluble Manganese — Official Final Action 
Atomic Absorption Spectrophotometry Method {48) 

2 . 1 39 Preparation of Sampie 

(a) Applicable to Mn +Z only. — Prep, sample soln as in 2.140, 
omitting the 50 mL H 3 P0 4 (1+9). Proceed as in 2.112, using std 
solns prepd as in 2.110(f) and (h), substituting 0.5/V H 2 S0 4 for 
0.5/V HCI in 2.110(h). 

(b) Applicable to total Mn+ 2 and Mn +A .~~ Prep, sample soln as 
in 2.111. Proceed as in 2.112, using std solns prepd as in 2.110(f) 
and (h). 

2.140 Coiorimetric Method {49) 

{Applicable to samples contg Mn +2 only and with ^5% Mn) 

Place 1 g sample in 200 mL wide-neck vol. flask or 250 mL 
beaker. Add 10 mL H 2 S0 4 and 30 mL HN0 3 . Heat gently until 
brown fumes diminish; then boil 30 min. If org. matter is not 
destroyed, cool, add 5 mL HN0 3 , and boil. Repeat process until 
no org. matter remains, and boil until white fumes appear. Cool 
slightly, and add 50 mL H 3 P0 4 (1+9). Boil few min. Cool, dil. to 
200 mL in vol. flask, mix, and let stand to allow pptn of CaS0 4 . 

Pipet 50 mL clear soln into beaker. Heat nearly to bp. With 
stirring or swirling, add 0.3 g KI0 4 for each 15 mg Mn present, 
and hold 30-60 min at 90-100°, or until color development is 
complete. Cool, and dil. to measured vol. that will provide 
satisfactory concn for coiorimetric measurement by instrument 
chosen (usually <20 ppm Mn). Compare in colorimeter against 
std KMn0 4 soln, 7.116, or in spectrophtr at 530 nm. Calc.'to Mn. 

2.141 * Bismuthate Method (50) * 

(Applicable to Mn +2 only) 
See 2.127-2.128, 11th ed. 



Water-Soluble Manganese 

2.142 Atomic Absorption Spectrophotometric 
Method {48)— Official Finai Action 

(Applicable to Mn +2 only) 

Place 1 g sample in 50 mL beaker, wet with alcohol, add 20 
mL H 2 0, and let stand 15 min, stirring occasionally. Transfer to 
9 cm Whatman No. 5 paper, and wash with small portions H 2 
until filtrate measures ca 230 mL, Let each portion pass thru 
paper before adding more. Add 3-4 mL H 2 S0 4 to filtrate. Proceed 
as in 2.112, using std solns prepd as in 2.110(f) and (h), 
substituting 0.5/V H 2 S0 4 for 0.5/V HCI in 2.110(h). 



Copper — Official Finai Action 
2.143 * Long Volumetric Method {51) * 
See 2.129-2.130, 11th ed. 



Short Volumetric Method (52) 



2.144 



Reagents 



(a) Sodium thiosulfate std sofn,~ -0.03/V. Prep, daily by dilg 
0.1/V soln, 50.037-50.038. 1 mL 0.03/V Na 2 S 2 3 = 1.906 mg Cu. 

(b) Starch soln. — Mix ca 1 g sol. starch with enough cold H 2 
to make thin paste, add 100 mL boiling H 2 0, and boil ca 1 min 
while stirring. 

(c) Bromocresoi green indicator. — Dissolve 0.1 g tetrabromo- 
/7?-cresolsulfonphthalein in 1.5 mL 0.1/V NaOH, and dil. to 100 
mL with H 2 0. 



AOAC Methods (1980) 



Other Elements 



25 



2.145 



Determination 



Place 2 g sample in 300 mL erlenmeyer and add 10 mL HN0 3 
and 5 mL H 2 S0 4 . Digest on hot plate to white fumes. If soln 
darkens, owing to org. matter, cool slightly, add little more 
HN0 3 , and digest again to white fumes, repeating operation if 
necessary until org. matter appears to be destroyed. Cool, add 
50 mL H 2 0, boil ca 1 min, and cool to room temp. 

Add bromocresol green, then NH 4 OH until indicator changes 
to light green (pH 4). Cool again to room temp., and if indicator 
changes back to more acid color, add NH 4 OH dropwise until 
indicator becomes light green again, avoiding excess. Add 2 g 
NH A HF 2 (Toxic. Caution: See 51.084), mix well, and let stand ca 
5 min. Add 8-10 g Kl, mix well, and titr. with std Na 2 S 2 3 soln to 
light yellow. Add ca 1 mL starch soln and continue titrg slowly 
until color is nearly same as just before addn of the Kl and 
becomes no darker on standing 20 sec. Report as % Cu. 

2.146 Atomic Absorption Method 
See 2.109-2.113. 

Sodium — Official First Action 
Flame Photometric Method [53)— Official First Action 

2.147 Reagents 

See 2.091(aMc), and in addn: 
Sodium chloride. — Dry 2 hr at 105°. 

2.148 Preparation of Solution 

Prep, soln as in 2.093(a), using 2.5 g sample (<4% Na) or 1.25 
g (4-20% Na). 

2. 1 49 Preparation of Standard Curve 

(a) Samples containing 1% or more sodium. — Proceed as in 
2.094, using 1.2716 g NaCI, range of dtln 0-40 ppm Na, intervals 
^5 ppm, and full scale for 40 ppm Na. 

(b) Samples containing less than 1% sodium. — Proceed as in 
2.094, using 1.2716 g NaCI, range of diln 0-10 ppm Na, intervals 
2 ppm, and full scale for 10 ppm Na. 

2.150 Determination 

{Caution: See 51.007.) 

Transfer 25 mL (<4% Na) or 10 mL (4-20% Na} sample soln 
to 250 mL vol. flask, dil. to vol. with H 2 0, and mix (if internal std 
instrument is used, add required amt LiN0 3 before dilg to vol.). 
Atomize portions of sample several times to obtain reliable av. 
readings for each soln. Det. ppm Na from std curve (a) or (b). 
Calc. % Na as follows: 

0-^1%: ppm Na/10 = % Na 
4-20%: ppm Na/2 - % Na 

Zinc — Official Final Action 

2.151 * Gravimetric Method [54) * 

(For samples contg ^0.1% Zn) 

Digestion with HN0 3 and H 2 S0 4 , sepn of interfering sulfides 
from acid soln with H 2 S, pptnofZnSatpH3with H 2 S, and ignition 
to ZnO. See 2.138, 12th ed. 

2.152 * Coiorimetric Method [55) • 

(For samples contg <4% Zn) 

Digestion with HN0 3 and H 2 S0 4 , and detn with dithizone. See 
2.139, 12th ed. 



Zincon Ion Exchange Method [56) 

(Clean all glassware with hot chromic acid or HN0 3 (1 + 1 ). Rinse 
thoroly with H 2 0. Caution: See 51.023, 51.026, and 51.030.) 

2.153 Reagents 

(a) Anion exchange resin. — 100-200 mesh, strong base, poly- 
styrene alkyl quaternary amine, 7% cross linkage, CGA-540 (J. 
T. Baker Chemical Co., No. 4602, or equiv.). 

(b) Zincon indicator. — Dissolve 0.12 g zincon (o-[[a-[(2~ 
hydroxy-5-sulfophenyl)azo]benzylidene]hydrazino]benzoicacid, 
Na salt) (J. T. Baker Chemical Co., No. X690) in 5 mL 0.3/V NaOH 
and dil. to 100 mL with H 2 0. Prep, fresh weekly. 

(c) Hydrochloric acid solns.—U) 0.5/V— Dil. 20 mL HCI to 500 
mL with H 2 0. (2) 0.25N.— Dil. 2 mL HCI to 100 mL with H 2 0. (5) 
0.005N.— Dil. 2.5 mL HCI to 6 L with H 2 0. 

(d) Borate buffer soln.— pH 9.8. Dissolve 4 g H 3 B0 3 in 140 mL 
H 2 0. Add 5 mL NH 4 OH by pipet and then dropwise to pH 9.8. 
Check daily. 

(e) Ammonium thiocyanate. — \M. Dissolve 0.76 g NH 4 CNS in 
10 mL H 2 0. 

(f) Zinc std solns.—{1) Stock soln. —1000 ppm. Dissolve 1.000 
g pure Zn metal in small amt HCI-HN0 3 (1+1). Evap. to small 
vol., add 3 mL HCI, and heat. Dil. to 1 L with H 2 0. (2) Working 
soln. — 10 ppm. Dil. 10 mL stock soln to 1 L with H 2 0. 

(g) Sodium hydroxide soln. — 0.3/V. Dissolve ca 1.25 g NaOH 
in100mLH 2 O. 

2. 1 54 Preparation of Resin Column 

Wash 12 g new resin in 250 mL beaker with H 2 until washings 
are neut. Introduce resin as slurry into 25 x 2.2 cm chromatgc 
tube with fritted glass disk and stopcock at bottom. Mark vol. 
levels on column at 10, 40, and 50 mL above packed resin and 
on 250 mL separator at 90 mL. (Keep resin wet and store under 
liq. when not in use.) Connect separator to top of column thru 
stopper. Attach inverted U-shaped glass dispensing tube to 250 
mL vol. flask thru vented stopper or cork and connect with Zn- 
free plastic tubing to stopcock of column with stopcock grease. 
See Fig. 2:08. 

Mount reservoir (aspirator bottle or carboy) contg 3*1 L 0.005/V 
HCI high enough to effect backwashing. Attach Zn-free tubing 
and pinch clamp. 



2.155 



Flow Calibration 



Use sweep sec hand of watch or stopwatch to establish flow 
rates. Det. number drops/mL leaving dispensing tube. Remove 
separator and vol. flask; drain and then backwash resin {see 
2.157). Remove reservoir tubing, open stopcock, elute 40 drops 
from dispensing tube, and measure vol. Use this factor to 
convert 0.5 mL/min (required in Zn elution, 2.157) to drops/ sec. 

2.156 Preparation of Sample 

Remove separator and elution tubing from column. Activate 
resin by draining column and adding 50 mL 0.5/V HCI. Drain 
column to 40 mL mark. 

(a) Samples containing 0. 14% or more zinc. — Dissolve 1.000 
g well-ground sample in 10 mL HCI and 5 mL HN0 3 in 250 mL 
beaker. Evap. to near dryness on hot plate. [Caution: Do not 
bake.) Redissolve residue in ca 40 mL 0.5/V HCI, boiling gently 
if necessary. Filter thru Whatman No. 41 paper into 100 mL vol. 
flask. Thoroly wash residue and dil. filtrate to vol. with 0.5/V HCI. 
Drain column to 10 mL mark. Tap column to pack resin. Pipet 
aliquot contg 0.7-0.8 mg Zn onto column. Elute sample soln at 
ca 5 sec/drop. 

if Surplus method— see inside front cover. 



26 



2. Fertilizers 



AOAC Methods (1980) 



VENTED 




2.158 



Determination 



FIG. 2:08— Apparatus for elution of resin column. 



(b) Samples containing less than 0.14% zinc. — Weigh, to 
nearest mg, sample contg 0.7-0.8 mg Zn into 250 mL beaker. 
Digest and filter sample and prep, column as in (a). Tightly 
attach open separator to column. Close stopcock. Transfer entire 
sample soln to separator, rinsing with two 10 mL portions 0.5/V 
HCI. Open stopcock. Elute sample soln at ca 5 sec/drop. Remove 
empty separator, rinse twice with 20 mL 0.5/V HCI, and add 
rinses to remaining soln in column. 



2.157 



Elution of Zinc 



After sample soln passes thru resin, immediately rinse column 
with 0.5/V HCI at ca 1.5 sec/drop until 1 mL eluate gives clear, 
colorless soln with 1M NH 4 SCN. If Fe +3 is present, soln will turn 
brown. Drain resin and backwash with 0.005/V HCI from reservoir 
thru elution tubing, forcing out air bubbles from tubing and 
column. Simultaneously, tap resin into suspension as it is forced 
up. Close column stopcock when liq. reaches 50 mL mark on 
column. Attach dispensing tube to vol. flask. Reopen stopcock, 
and raise flask until flow just stops. Continue ht adjustment until 
a drop remains in equilibrium at tip of dispensing tube and 
neither rises nor falls. Secure flask. Attach open separator with 
H 2 seal to column and close stopcock. Add 240 mL 0.005/V HCI 
to separator and reopen stopcock. Open column stopcock until 
rate of ca 0.5 mL/min is sustained 10 min. If rate decreases, 
increase rate slightly until nearly const. Let elution continue 
overnight. Then, if >90 mL remains in separator, readjust rate 
as above and continue elution to 90 mL mark. Finally, lower 
flask, fill to 250 mL mark at convenient rate from dispensing tip, 
and mix. Detach hose and separator from column, and drain all 
3. Reactivate resin, and stopper column as in 2.156. 



Pipet 20 mL eluate into 50 mL vol. flask contg small piece 
litmus paper. Make alk. with 0.3/V NaOH, then just acidic with 
0.25/V HCI. Pipet in 2 mL more acid, 5 mL buffer, and 3 mL 
zincon soln. Dil. to vol. with H 2 0. Similarly prep. 0, 1, 2, and 3 
ppm std solns, using 0, 5, 10, and 15 mL std Zn working soln, 
resp. Using ppm std soln as blank, det A at 620 nm 1 5-45 min 
after zincon addn. Plot std curve of ppm against A. 

% Zn = <C x F)/W, 
where C = ppm from std curve; W = g sample; and F = 0.0625 
for samples contg <0.14% Zn or 6.25/mL aliquot pipetted onto 
resin for samples contg 2=0.14% Zn. 

2.159 Atomic Absorption Method 
See 2.109-2.113. 



Total Sulfur (57) — Official Final Action 
2.160 Reagents 

(a) Barium chloride soln.— 10%. Dissolve 100 g BaCI 2 .2H 2 in 
900 mL H 2 and filter thru Whatman No. 42 paper, or equiv. 1 
mL = 14 mg S. 

(b) Bromine in carbon tetrachloride. — 10%. Add 10 g Br to 90 
g reagent grade CCI 4 . Stir until homogeneous. Store in g-s bottle. 
{Caution: See 51.047 and 51.049.) 



2.161 



Determination 



Weigh sample contg 50-150 mg S into 250 mL beaker, and 
add 20 mL 10% Br in CCI 4 , (b). Mix by swirling beaker at 5 min 
intervals during 30 min. Add 15 mL HN0 3 and mix as before. 
Evap. to 1-2 mL on warm hot plate. Add 15 mL HCI and 10 mL 
H 2 0. Evap. just to dryness on warm hot plate or steam bath. 
Add 10 mL HCI and 50 mL H 2 0, heat to boiling, boil 5 min, and 
filter thru Whatman No. 42 paper, or equiv. Wash paper with 
ten 20 mL portions hot H 2 0. 

Heat filtrate to boiling. Add 5-6 drops 10% BaCI 2 soln, (a). 
After 1 min, add dropwise amt BaCI 2 soln equiv. to expected S 
content plus 5 mL excess. Digest at gentle boil 1 hr. Remove 
from hot plate and let ppt settle 15-20 min. Filter immediately 
thru previously ignited and weighed gooch. Wash with hot H 2 
until 10 mL wash H 2 shows no ppt with 3 mL 1% AgN0 3 . Dry 
and ignite at 800° to const wt. Cool in desiccator over MgCI0 4 
and weigh. 

% S = g BaS0 4 x 0.1374 x 100/g sample. 

2.162 Free Sulfur (55)— Official Final Action 

{Caution: See 51.011, 51.047, 51.048, and 51.049.) 

Ext 1 g sample with CS 2 in Soxhlet app., letting extn thimble 
drain s*12 times. Transfer ext to 250 mL beaker. Evap. CS 2 in 
draft at room temp. Heat in oven 20 min at 60-70°; then cool to 
room temp. Add 10 mL satd soln of Br in CCI 4 , cover, and let 
stand ca 30 min, stirring several times. Add 15 mL HN0 3 , cover, 
and let stand ca 30 min, stirring several times. Evap. on hot 
plate to ca 5 mL. Add 20 mL HCI and evap. to ca 5 mL. Add ca 
50 mL H 2 0, filter, and wash with HCI (1+49). Add 2 drops 
bromophenol blue, 6.019(f), an^ then NH 4 OH to first color 
change. Add HCI dropwise until distinctly acid, then 5 drops 
excess; dil. to 150 mL, heat to bp, and add 10% BaCI 2 soln, 
2.160(a), dropwise until ca 50% excess is present. Cover beaker 
and digest on steam bath ^=1 hr. Cool to room temp, and filter 
thru asbestos on gooch previously ignited at 800° and weighed. 
Wash 10 times with hot H 2 0. Ignite in furnace at 800° ^20 min. 
Cool in desiccator and weigh as BaS0 4 . Calc. as S as in 2.161. 



AOAC Methods (1980) 



Peat 



27 



2.163 if Acid-Forming or Nonacid-Forming * 

Quality (55)— Official Final Action 

Fusion with std amt Na 2 C0 3 followed by neutzn of melt with 
excess acid and back-titrn with std NaOH. See 2.141-2.142, 11th 
ed. 

PEAT 

(Moss, humus, and reed-sedge types) 

2.164 Sampling (50)— Procedure 

Use slotted single or double tube or slotted tube and rod, all 
with pointed ends and min. 1" diam. for loose materials. Use 
cutting type core sampler, with plunger, for compressed ma- 
terials. Pennsylvania State Forage Sampler (NASCO, 901 Janes- 
ville Ave, Fort Atkinson, Wl 53538) is satisfactory core sampler. 

Take representative sample from lot or shipment as follows: 

(a) Packaged or baled peats,— Lay bag or bale horizontally 
and remove core diagonally from end to end. From lots of 1-10 
bags, sample all bags; from lots of s*11, sample 10 bags. Take 
1 core from each bag sampled; except for lots of 1-4 bags, take 
diagonal cores from each bag and addnl cores to total 5=5 cores. 

(b) Bulk samples. — Draw s*10 cores from different regions. 

(c) Small containers {10 lb or less). — Take entire package. 
Working rapidly to prevent moisture losses, reduce composite 
sample to =£500 g (by wt) or 2 L (by vol.) by mixing on clean 
plastic or paper and quartering. Place sample in air-tight con- 
tainer. 

Sampling by random "grab" procedure is necessary if particle 
size range is to be detd or if representative sample cannot be 
taken with core sampler as above. 

2.165 Preparation of Sample {67)— Official Final Action 

Place representative field sample on square rubber sheet, 
paper, or plastic. Reduce sample to amt required by quartering 
and place in moisture-proof container. Work rapidly to prevent 
moisture losses. 

Moisture [61) — Official Final Action 

2.166 Method I 

Mix sample thoroly and place 10-12 g in ignited and weighed 
(with fitted heavy-duty Al foil cover) Vycor or porcelain evapg 
dish, =*75 mL capacity. Crush soft lumps with spoon or spatula. 
Cover immediately with Al foil cover and weigh to nearest mg. 
Dry, uncovered, 16 hr at 105°. Remove from oven, cover tightly, 
cool, and weigh. 

% Moisture (report to nearest 0.1%) = (g as-received sample 
- g oven-dried sample) x 100/g as-received sample. 

2.167 Method II 

(Use when pH, N, fiber, etc., are to be detd.) 

Mix thoroly and weigh 100-300 g representative sample, 
2.165, and spread evenly on large flat pan. Crush soft lumps 
with spoon or spatula and let come to moisture equilibrium with 
room air 3=24 hr. Stir occasionally to maintain max. air exposure 
of entire sample. When wt is const calc. loss in wt as % moisture 
removed by air drying. Grind representative portion air-dried 
sample 1-2 min in high-speed blender; use for moisture, ash, 
and N detns. 

Mix air-dried, ground sample and weigh, to nearest mg, equiv. 
of 10 g sample on as-received basis (g air-dried sample equiv. 



* Surplus method— see inside front cover. 



to 10.0 g as-received sample = 10.0 - [(10.0 x % moisture 
removed)/ 100]). Place weighed sample in ignited and weighed 
(with fitted heavy duty Al foil cover) Vycor or porcelain evapg 
dish and proceed as in 2.166. 

% Moisture (report to nearest 0.1%) = (10.0 - g oven-dried 
sample) x 10.0. 

Mechanical Analysis for Determination of 
Particle Size Range {60) — Procedure 



2.168 

Mechanical sieve shaker. — With 8" diam. 
equipped with cover and bottom pan. 



Apparatus 

, Nos. 8 and 20 sieves 



2.169 

Air-dry as in 2.167. 

2.170 



Preparation of Sample 



Determination 

Mix thoroly and place 20.0 g air-dried sample on No. 8 sieve 
nested on No. 20 sieve. Secure sieves and shake at suitable 
speed 10 min. Remove and weigh foreign matter, such as sticks, 
stones, and glass, from No. 8 fraction. Weigh fractions of peat 
retained on Nos. 8 and 20 sieves and portion collected in bottom 
pan. Convert fraction and sample wts to as-received basis and 
calc. in terms of %. (If foreign matter is absent, conversion to 
as-received basis is not necessary.) 

% Foreign matter - fraction removed from No. 8 sieve x 100; 

% Coarse fiber = fraction retained on No. 8 sieve x 100; 

% Medium fiber = fraction retained on No. 20 sieve x 100; 

% Fine - fraction collected in pan x 100. 

If mech. sieve shaker is not available, use hand sieving. 
Conduct sieving by appropriate lateral and vertical motions 
accompanied by jarring action. Continue until no appreciable 
change is noted in sieve fraction. 



pH {60) — Procedure 



2.171 



Apparatus and Reagents 



(a) pH meter. — Battery-operated or on elec. line with voltage 
regulator. 

(b) Carbon dioxide-free water.— See 50.007. 

(c) Acid potassium phthalate buffer soin. — 0.05m. See 
50.007(c). 

(d) Phosphate buffer soln.-— O.Olbm. See 50.007(d). 

(e) Calcium chloride solns {Method If only). — ( 7 ) Stock soln. — 
1 .0M. Dissolve 147 g CaCI 2 .2H 2 in H 2 in 1 L vol. flask, cool, dil. 
to vol., and mix. Dil. 15 mL of this soln to 200 mL with H 2 in 
vol. flask and stdze by titrg 25 mL aliquot dild soln. with std 
0.1/V AgN0 3 , 50.029, using 1 mL 5% K 2 Cr0 4 as indicator. {2) 
Working sofn.—Q.QlM (pH 5.0-6.5). Dil. 20 mL stock soln. to 2 
L with H 2 0. 



2.172 



Determination 



(a) Method I [in distilled water). — Weigh ca 3.0 g air-dried 
peat or equiv. amt moist material into 100 mL beaker. Add 50 
mL H 2 0. (Addnl H 2 may be needed for very fibrous materials 
such as sphagnum moss peat.) Let soak 30 min, with occasional 
stirring. Read on pH meter. 

(b) Method II {in 0.01 M calcium chloride soln). — Weigh ca 
3.0 g air-dried peat or equiv. amt moist material into 100 mL 
beaker. Add 50 mL 0.01M CaCI 2 . Let soak 30 min, with occasional 
stirring. Read on pH meter. Report results as pH in 0.01M CaCI 2 
soln. (pH values in CaCI 2 soln. are usually ca 0.5-0.8 units lower 



28 



2. Fertilizers 



AOAC Methods (1980) 



than those in H 2 0. Observed pH in CaCI 2 soln is virtually 
independent of initial amt salt present in soil, whereas pH 
readings in H 2 can be modified by salts such as fertilizer 
material.) 

2.173 Ash (fi/)— Official Final Action 

Place uncovered (retain cover for weighing) Vycor or porcelain 
dish contg dried sample from moisture detn in furnace. Gradually 
bring to 550° and hold until completely ashed. Cover with 

retained Al foil cover, cool, and weigh. 

% Ash (report to nearest 0.1%) = g ash x 100/g as-received 
sample taken for moisture detn, (If moisture Method II was used, 
g as-received sample = 10.0.) 

Sand [60) — Procedure 

2.174 Preparation of Sample 

Air-dry as in 2.167. 



2.175 



Determination 



{Caution: See 51.056.} 



Place 25 g air-dried, ground sample into 125 mL tall-form 
beaker, or equiv. Nearly fill beaker with CHCI 3 , stir briefly, and 
let settle ca 1 min. With spoon, discard most floating org. 
material, decant remaining org. material and CHCI 3 , taking care 
not to disturb settled portion (sand), and air-dry to remove 
residual CHCI 3 . (Stirring aids drying.) 

When dry, weigh settled portion and catc. as % sand (includes 
other minerals present such as limestone, etc.). 

%Sand = (g air-dried settled residue x 100)/g air-dried sample 

2.176 Organic Matter (57)— Official Final Action 

% Org. matter = 100.0 - (% moisture + % ash). 

2.177 Total Nitrogen (6Y>)-~Procedure 

Det. N as in 2.057, using well mixed, air-dried, ground sample 
equiv. to 10.0 g sample on as-received basis. 

Det. g air-dried sample equiv. to 10.0 g as-received sample as 
in 2.167. 

Water Capacity and Volumes 162) 
Official Final Action 

2.178 Apparatus 

Dispensing apparatus. — 2 dispensing burets, 250 mL in 1 mL 
subdivisions, ±2 mL tolerance, pinchcock type; 1-hole No. 6 
rubber stopper; straight polyethylene drying tube with serrated 
rubber tubing fittings, 15 cm long, %" od, 5 / 8 " id (Cenco Instru- 
ment Corp. No. 14782-2); and stainless steel screen circle, ca 
16 mesh and 28.7 mm diam. 

Assemble dispensing app. as follows: Discard serrated rubber 
tubing fittings from polyethylene drying tube and use tube only. 
Center stainless steel screen on one end of tube and seal. 
(Soldering iron is useful.) Adjust length of tube to match con- 
venient graduation of buret; then scallop end without screen to 
allow for H 2 drainage, and insert into dispensing buret with 
screen side up. 



2.179 

See 2.165. 



Preparation of Sample 



Weigh buret fitted with plastic tube and screen. Working 
rapidly to prevent moisture losses, mix sample thoroly, place 
on top of No. 4 screen, and shake until sieving is complete. Use 
only portion that has passed thru sieve for detn. Firmly pack 
buret with 25 cm (10") of 4 mesh sample as follows: Attach 
rubber stopper to delivery end of buret. Add ca 20 mL portions, 
firmly tapping 3 times vertically from ht of 15 cm (6") on rubber 
stopper, for final ht of 25 cm. (This will ensure that ht of final 
wet vol. is 19-25 cm.) Remove stopper; weigh buret to nearest 
g. Position buret to use sink as drain. Place H 2 source (19 L 
(5 gal.) bottle) equipped with siphon device above level of buret. 
Connect clamped rubber tubing of siphon device to buret with 
glass tubing (ca 13 cm (5") long, constricted at one end) inserted 
into one-hole rubber stopper fitting tightly into top of buret. 
Attach rubber tubing with pinch clamp to delivery end of buret. 
Open both clamps and pass H 2 thru sample s=24 hr, maintaining 
water reservoir over sample at all times. (Moss-type samples 
may float but gradually settle as sample becomes wet.) After 
initial soaking, regulate H 2 flow thru column by adjusting screw 
clamp at delivery end of buret. (In-flow of H 2 should be ca 
equal to out-flow; flow of ca 1 drop/sec is suitable.) When 
sample is supersatd, close both clamps and let sample settle in 
H 2 ca 5 min. Top surface of sample should be as level as 
possible. 

Raise buret and replace rubber tubing on delivery end of buret 
with 250 mL dispensing buret filled with H 2 0, using rubber 
stopper for connection. Connect two burets tightly, with no air 
leaks. Remove siphon device and open outlet clamps of both 
burets to empty. (Suction created is equiv. to ca 38 cm (15") H 2 0. 
Check for air leaks to ensure that std suction is exerted on 
sample. It is important to remove excess H 2 as described.) 
Measure ht of wet peat. Ht should be 19-25 cm. Record vol. in 
mL and weigh buret, plastic tube with screen, and wet peat to 
nearest g. 

Wet sample again as above ^1 hr, drain by suction, record 
vol., and weigh. Repeat until consistent results are obtained. 



2.181 



Calculations 



(a) Saturated Volume Weights, g/mL 
As-recd = g as-recd sample/mL wet vol. 
Oven-dried = g dried sample/mL wet vol., where g dried 
sample = g as-recd sample x [(100 - % moisture)/ 100]. 
Wet = g wet sample/mL wet vol. 

(b) Water-Holding Capacity, % 
(7) Weight basis: 

As-recd - [(g wet sample - g as-recd sample) x 100]/g 

as-recd sample 
Oven-dried = [(g wet sample - g dried sample) x 100]/g 
dried sample 
(2) Volume basis: 

Watervol. = [(g wet sample - g dried sample) x 100]/(mL 
wet vol. x 1.0) 
(c) Dry Peat Volume, % 
Dry peat vol. = (g dried sample x 100)/(mL wet vol. x1.5) 

(d) Air Volume, % 
Air vol. - 100 - (% water vol. + % dry peat vol.) 



Alternative Methods (60) 
Volume — Procedure 



2.182 



Principle 



2. 1 80 Determination 

Det. moisture content on sep. sample by 2.166 or 2.167. 



Method consists of dividing particles of peat from original 
container by passing them thru 1 2.7 mm (0.5") sieve and allowing 
them to fall into vol, -measuring container. 



AOAC Methods (1980) 



Peat 



29 



2.183 Apparatus 

(a) Sieve.— No. V 2 " (12.7 mm). 

(b) Measuring box. — Steel or wood, bound with metal having 

one of the following sets of inner dimensions: (7) V 2 cu. ft. 

= 12x12x12" with line scribed 6" from bottom; {2) V* cu. ft. 

= 12 x 12 x 12" with line scribed 9" from bottom; (3) 1 cu. ft. 

- 12 x 12 x 12" box, 2 cu. ft. = 16 x 16 base x 13.5" ht, 5 cu. 

ft - 16 x 16 base x 33.75" ht. 



2.184 



Determination 



(a) Loose peat — Remove material from bag or container, 
pass it thru !/ 2 " sieve, and place directly into measuring box. 
Pour contents from ca 60 cm (2') into measuring box. Det. 
contents of bag or container only once. Fill corners of measuring 
box by shaking with rotary motion, 1 rotation/sec for 5 sec, 
without lifting box from floor or surface. When filled, level off 
by straightedge. Use ht of box to calc. vol. in cu. ft. 

(b) Bated peat. — Vol. baled material = ht x area of base. Cor- 
rect measurements for outside wrappers. Det. amt loose peat 
in bale by passing thru Va" sieve and measuring amt loose peat, 
using 12 x 12 x 12" box as in (a). Report vol. of peat in cu. ft. 

Report total vol. of sieved peat from original container. 

Volume Weight, Water-Holding Capacity, 

and Air Capacity of Water-Saturated 

Peat Materials — Procedure 

2.185 Apparatus 

(a) Hoi low spray nozzle.— Monarch F-97-W, nozzle No. 4.6160 
{Monarch Mfg. Works Inc., 2501 E Ontario St, Philadelphia, PA 
19134), or equiv. 

(b) Pipe connection. — For installation of nozzle on H 2 faucet 
in sink. 

(c) Containers. — Approx. 2 L (2 lb coffee cans are suitable) 
fitted with plastic covers. Replace metal bottom of one with No. 
20 Cu screen (test container). 

(d) Aluminum pie pans. — 20 cm (8") diam. Drill holes in side 
walls of pan so that H 2 depth in pan remains ca 1.3 cm (0.5"). 



2.186 
See 2.165. 

2.187 



Preparation of Sample 



Determination 

Det. moisture content on sep. sample by 2.166 or 2.167. 

Weigh test container fitted with plastic cover, screen, and 
circle of filter paper (12.5 cm Whatman No. 4, or equiv.) which 
is placed on screen. Thoroly mix equal wts of H 2 and peat and 
place in container without pressure to ht of 10 cm (4"); record 
wt in g. (If peat is dried out, mix 1 part peat with 2 parts H 2 0. 
If wet, mix 2 parts peat with 1 part H 2 0.) 

Place test container in Al pan filled with H 2 in sink s=30 cm 
(12") directly under spray nozzle. Water ca 24 hr as mist to 
prevent compression of peat. Place cover on container, seal 
(tape is suitable) to prevent evapn, and let stand in Al pan, 
maintaining 1.3 cm H 2 head 2 days. Remove from pan and 
drain 2 hr with container at 45° angle. Remove seal on cover, 
and record wt and vol. Vol. can be detd by using container 
identical to test container not fitted with screen, filling H 2 to 
same ht as sample in test container, and transferring to graduate 
with mL markings. 



2.188 



Caicuiations 



sample = g total sample/2; or g total sample/3 if 2 parts H 2 
used; or (g total sample x 2)/3 if 2 parts peat used. 
(2) Oven-dried.— See 2.181. 
{3) Wet— See 2.181. 

(b) Water-Holding Capacity, % 
See 2.181. 

(c) Dry Peat Volume, % 
See 2.181. 

(d) Air Volume, % 
See 2.181. 



Cation Exchange Capacity {63) 
Official Final Action 

AOAC-ASTM Method 
2.189 Principle 

Cation exchange capacity is measure of total amt exchange- 
able cations that can be held by peat, expressed as mequiv./lOO 
g air-dried peat. Peat sample is shaken with 0.5/V HCI to remove 
bases and to sat. sorption complex with H + . Excess acid is 
removed; absorbed H + is replaced with Ba +2 , titrd with 0.1/V 
NaOH, using phthln indicator, and calcd to mequiv./100 g air- 
dried peat. 



2.190 



Reagents 



(a) Dilute hydrochloric acid.— 0.5/V. Dil. 42 mL HCI to 1 L with 
H 2 0. 

(b) Barium acetate soln. — 0.5/V. Dissolve 64 g Ba(OAc) 2 in H 2 
and dil. to 1 L 

(c) Silver nitrate soln. — 1%. Dissolve 1 g AgN0 3 in 100 mL 
H 2 0. 

(d) Sodium hydroxide std soln. — 0.1/V. Prep, and stdze as in 
50.032-50.035. 



2.191 
See 2.165. 

2.192 



Preparation of Sampie 



Determination 



(a) Saturated Volume Weights, g/mL 
(/) As-recd = g as-recd sample/mL wet vol., where g as-recd 



Thoroly mix air-dried ground peat sample and place 2.00 g in 
300 mL erlenmeyer. Add ca 100 mL 0.5/V HCI; stopper flask and 
shake vigorously periodically during 2 hr (or shake mech. 30 
min). Filter thru rapid paper (24 cm fluted, or equiv.) in large 
powder funnel. Wash with 100 mL portions H 2 until 10 mL 
wash shows no ppt with ca 3 mL 1% AgN0 3 . Discard filtrate. 
Immediately transfer moist peat to 300 mL erlenmeyer, by 
puncturing apex of paper and forcing moist peat thru funnel 
stem into erlenmeyer, using spray from wash bottle contg ca 
100 mL 0.5/V Ba(OAc) 2 . Stopper flask and shake vigorously 
periodically during 1 hr (or shake mech. 15 min). Filter, and 
wash with three 100 mL portions H 2 0. Discard peat, and tttr. 
washings with 0.1/V NaOH, using 5 drops phthln, to first pink. 

Calc. mequiv./100 g air-dried peat = (mL x normality 
NaOH x 100)/g sample. 

SELECTED REFERENCES 

(/) JAOAC 12, 97(1929); 33, 424(1950); 38, 108, 541(1955); 50, 

190, 382(1967); 51, 859(1968); 55, 709(1972). 
(2) JAOAC 52, 592(1969). 
(5) JAOAC 42, 500(1959). 

(4) JAOAC 12, 98(1929); 24, 253(1941). 

(5) JAOAC 3, 95(1917). 
{6) JAOAC 40, 711(1957). 



30 



2. Fertilizers 



AOAC Methods (1980) 



(7) JAOAC 46, 582(1963); 47, 32, 1040(1964). 

(8) JAOAC 52, 1 127(1969); 55, 699(1972), 

(9) JAOAC 38, 413(1955). 

(10) JAOAC 41, 517(1958); 42, 503(1959). 

(11) Z. Anal. Chem. 189, 243(1962); JAOAC 45, 40, 201, 
999(1962); 46, 579(1963); 47, 420(1964). 

(12) Z. Anal. Chem. 189, 243(1962); JAOAC 45, 40, 999(1962); 
49, 1201(1966); 52, 587(1969). 

(13) JAOAC 61, 533(1978). 

(14) JAOAC 5, 443, 460(1922); 6, 384(1923); 14, 182(1931); 19, 
269(1936); 22, 254(1939); 42, 512(1959). 

US) JAOAC 42, 503(1959). 

(16) JAOAC 52, 587(1969). 

(17) JAOAC 43, 478(1960); 46, 570(1963); 60, 702(1977). 

(18) JAOAC 44, 233(1961). 

(19) JAOAC 46, 570(1963); 47, 420(1964). 

(20) JAOAC 38, 56(1955). 

(21) JAOAC 53, 450(1970); 57, 10(1974). 

(22) JAOAC 61, 299(1 978). 

(23) Chem. Ztg. 16, 1952(1892); JAOAC 6, 391(1923); 15, 
267(1932). 

(24) JAOAC 13, 208(1930); 15, 267(1932). 

(25) JAOAC 18, 62, 218(1935); 19, 68, 279(1936). 

(26) JAOAC 53, 808(1970); 56, 853(1973). 

(27) JAOAC 13,215(1930). 

(28) JAOAC 38, 436(1955); 44, 245(1961). 

(29) Ind. Eng. Chem., Anal. Ed. 7, 259(1935); JAOAC 41, 
637(1958); 42, 494(1959). 

(30) JAOAC 43, 499(1960); 57, 1360(1974); 59, 22(1976); 60, 
323(1977). 

(37) JAOAC 59, 22(1976). 

(32) JAOAC 18, 237, 260, 281(1935); 19, 302(1936). 

(33) Anal. Chem. 21, 984(1949); JAOAC 35, 674(1952); 36, 
649(1953). 

(34) JAOAC 28, 782(1945). 



(35) J. Agrtc. Food Chem. 3, 48(1955); JAOAC 41, 533(1958); 
51,857(1968). 

(36) JAOAC 53, 456(1970); 54, 646(1971). 

(37) Anal. Chem. 29, 1044(1957); 30, 1882(1958); JAOAC 41, 
533(1958); 43, 472(1960). 

(38) JAOAC 52, 566(1969). 

(39) JAOAC 48, 406, 1100(1965); 50, 401(1967); 51, 847(1968); 
58,928(1975). 

(40) JAOAC 32, 422(1949); 33, 132(1950); 36, 623(1953); 38, 
407(1955). 

(41) JAOAC 38, 413(1955). 

(42) JAOAC 11, 34, 201(1928); 16, 69(1933). 

(43) JAOAC 24, 302(1941). 

(44) Anal. Chem. 30, 1153(1958); JAOAC 48, 412(1965). 

(45) JAOAC 50, 397(1967). 

(46) JAOAC 47, 450(1964). 

(47) JAOAC 20, 252(1937); 22, 270(1939); 23, 249(1940); 24, 
268(1941); 25, 326(1942). 

(48) JAOAC 55, 695(1972). 

(49) JAOAC 23, 249(1940). 

(50) JAOAC 24, 268(1941). 

(51) JAOAC 24, 305(1941). 

(52) JAOAC 25, 77, 352(1942). 

(53) JAOAC 55, 986(1972); 56, 859(1973); 57, 1402(1974). 

(54) JAOAC 25, 77, 361(1942). 

(55) JAOAC 25, 78(1942). 

(56) JAOAC 56, 846(1973). 

(57) JAOAC 47, 436(1964). 

(58) JAOAC 25, 348(1942). 

(59) JAOAC 19, 284(1936); 22, 289(1939). 

(60) Book of ASTM Stds (1971) Pts 1 1, 22, and 30, ASTM D2973- 
D2978, D2980, and D2944; JAOAC 56, 154(1973). 

(61) JAOAC 50, 394(1967). 

(62) JAOAC 51, 1296(1968); 52, 384(1969). 

(63) JAOAC 56, 154(1973). 



3. Plants 



3.001 Sampling (/) — Official Final Action 

When more than one plant is sampled, include enough plants 
in sample to ensure that it adequately represents av. composition 
of entire lot of plants sampled. {This number depends upon 
variability in composition of the plants.) Det. details of sampling 
by purpose for which sample is taken. 

3.002 Preparation of Sample (7) — Official Final Action 

(a) For mineral constituents. — Thoroly remove all foreign 
matter from material, especially adhering soil or sand, but to 
prevent leaching, avoid excessive washing. Air- or oven-dry as 
rapidly as possible to prevent decomposition or wt loss by 
respiration, grind, and store in tightly stoppered bottles. If results 
are to be expressed on fresh wt basis, record sample wts before 
and after drying. When Cu, Mn, Zn, Fe, Al, etc. are to be detd, 
avoid contaminating sample by dust during drying and from 
grinding and sieving machinery. 

(b) For carbohydrates. — Thoroly remove all foreign matter 
and rapidly grind or chop material into fine pieces. Add weighed 
sample to hot redistd alcohol to which enough pptd CaC0 3 has 
been added to neutze acidity, using enough alcohol so that final 
concn, allowing for H 2 content of sample, is ca 80%. Heat 
nearly to bp on steam or H 2 bath 30 min, stirring frequently. 
(Samples may be stored until needed for analysis.) 

3.003 Moisture — Official Final Action 
See 7.003, 7.006*, or 7.007. 

3.004 Ash— Official Final Action 
See 31.012, 31.013, or 7.009. 

3.005 Sand and Silica — Official Final Action 

Ignite 10-50 g sample in flat-bottom Pt dish in furnace, at 
500-550°, until residue is white or nearly so. {Use Pt dishes with 
caution in ashing plant materials high in Fe; for such materials, 
use well-glazed porcelain crucibles and include blank detn.) 
Moisten with 5-10 mL HCI, boil ca 2 min, evap. to dryness, and 
heat on steam bath 3 hr to render Si0 2 insol. Moisten residue 
with 5 mL HCI, boil 2 min, add ca 50 mL H 2 0, heat on H 2 bath 
few min, filter thru hardened paper, and wash thoroly. To this 
filtrate add filtrate and washings from alkali-sol. Si0 2 detn (b) 
and dil. to 200 mL. Designate as Soin /. 

(a) Sand. — Wash residue from filter into Pt dish and boil ca 5 
min with ca 20 mL satd Na 2 C0 3 soln; add few drops 10% NaOH 
soln, let settle, and decant thru ignited and weighed gooch. Boil 
residue in dish with another 20 mL portion Na 2 C0 3 soln and 
decant as before. Repeat process. Transfer residue to gooch and 
wash thoroly, first with hot H 2 0, then with little HCI (1+4), and 
finally with hot H 2 until Cl-free. Dry filter and contents, ignite 
at 500-550°, and weigh as sand. Confirm by microscopic ex- 
amination. 

(b) Alkali-soluble Si0 2 . — Combine a Ik. filtrate and washings 
from (a), acidify with HCI, evap. to dryness, add 5 mL HCI, again 
evap., and dehydrate by heating 2 hr at 110-120°. Moisten 
residue with 5-10 mL HCI, boil ca 2 min, add ca 50 mL H 2 0, and 
heat on H 2 bath 10-15 min. Filter thru ashless filter or ignited 
and weighed gooch, wash with hot H 2 0, ignite at 500-550°, and 
weigh as Si0 2 . Add filtrate to Soln I. 



METALS 

Calcium, Copper, Iron, Magnesium, 
Manganese, Potassium, and Zinc 

Atomic Absorption Method (2)— Official First Action 

3.006 Apparatus and Reagents 

Deionized H 2 may be used. See 2.109-2.110, and following: 

(a) Potassium stock soln .— 1000 m9 K/mL Dissolve 1.9068 g 

dried (2 hr at 105°) KCI in H 2 and dil. to 1 L. Use following 

parameters for Table 2:04: 7665 A, air-C 2 H 2 flame, and 0.04-2 

/u-g/mL range. 



3.007 



Preparation of Sample 



(a) Dry ashing. — Accurately weigh 1 g sample, dried and 
ground as in 3.002(a), into glazed, high-form porcelain crucible. 
Ash 2 hr at 500°, and let cool. Wet ash with 10 drops H 2 0, and 
carefully add 3-4 mL HN0 3 (1 + 1). Evap. excess HN0 3 on hot 
plate set at 100-120°. Return crucible to furnace and ash addnl 
1 hrat 500°. Cool crucible, dissolve ash in 10 mL HCI (1 + 1), and 
transfer quant, to 50 mL vol. flask. 

(b) Wet ashing. — Accurately weigh 1 g sample, dried and 
ground as in 3.002(a), into 150 mL Pyrex beaker. Add 10 mL 
HN0 3 and let soak thoroly. Add 3 mL 60% HCI0 4 and heat on 
hot plate, slowly at first, until frothing ceases. {Caution: See 
51.019.) Heat until HN0 3 is almost evapd. If charring occurs, 
cool, add 10 mL HN0 3 , and continue heating. Heat to white 
fumes of HCI0 4 . Cool, add 10 mL HCI (1 + 1), and transfer quant, 
to 50 mL vol. flask. 

3.008 Determination 

To soln in 50 mL vol. flask, add 10 mL 5% La soln, and dil. to 
vol. Let silica settle, decant supernate, and proceed as in 2.112. 

3.009 Calculations 

ppm Element - (^g/mL) x F/g sample, 
% Element = ppm x 10"* . 
where F - (mL original diln x mL final diln)/mL aliquot if 
original 50 mL is dild. 

3.010 Iron and Aluminum [3) — Official Final Action 

(Caution: See 51.030.) 

Take aliquot of Soln /, 3.005, contg enough Fe and Al to form 
ca 40 mg Fe- and AIP0 4 . Add few drops HN0 3 , Br-H 2 0, or H 2 2 
to oxidize Fe. If soln does not already contain excess phosphate, 
add 0.5 g (NH 4 ) 2 HP0 4 , stir until dissolved, anddiL.to 50 mL with 
H 2 0. Add few drops thymol blue soln, 22.040(e), and then add 
NH4OH until soln just turns yellow. Add 0.5 mL HCI and 25 mL 
25% NH 4 OAc soln, and stir. Let stand at room temp, until ppt 
settles (ca 1 hr). Filter, and wash 10 times with hot 5% NH 4 N0 3 
soln. Ignite at 500-550° and weigh as FeP0 4 and AIP0 4 . 

Fuse ignited residue in Pt crucible with ca 4 g Na 2 C03-K 2 C0 3 
(1+1) mixt. When fusion is complete, let crucible cool, add 5 mL 
H 2 S0 4 , and heat until copious fumes of S0 3 are evolved. Cool, 
transfer to flask, add H 2 0, and digest until soln is clear. Reduce 
Fe with Zn, cool, and titr. with 0.1/V KMn0 4 . Correct for blank 
and calc. as % Fe or % Fe 2 3 . Calc. to FeP0 4 and subtract from 
total Fe- and AIP0 4 to obtain AIP0 4 . Correct for blank and report 
as AI2O3. 



31 



32 



3. Plants 



AOAC Methods (1980) 



Methods for Iron Only 
Colorimetric Method (4) — Official Final Action 

3.011 Reagents 

(a) Acetic acid.—2M. Dil. 120 g HOAc to 1 L with H 2 0. 

(b) Ammonium citrate soln. — 1%. Dissolve 1 g NH 4 citrate in 
H 2 and dil. to 100 mL. 

(c) Bromophenol blue indicator. — 0.04%. Grind 0.1 g bromo- 
phenol blue in mortar with 3 mL 0.05/V NaOH, transfer to vol. 
flask, and dil. to 250 mL with H 2 0. 

(d) Buffer so/ns.—(1) pH 3.5.— Mix 6.4 mL 2M NaOAc with 
93.6 mL 2M HOAc and dil. to 1 L (2) pH 4.5.— Mix 43 mL 2/W 
NaOAc with 57 mL 2M HOAc and dil. to 1 L 

(e) Hydroquinone soln. — Dissolve 1 g hydroquinone in 100 
mL pH 4.5 buffer, (d)(2). Keep in refrigerator, and discard when 
any color develops. 

(ff) o-Phenanthroline soln. — Dissolve 1 g o-phenanthroline 
H 2 in H 2 0, warming if necessary, and dil. to 400 mL. 

(g) Sodium acetate soln.— 2M. Dissolve 272 g Na0Ac.3H 2 in 
H 2 and dil. to 1 L 

(h) Iron std soln. — 1 mg/mL. Dissolve 1 g electrolytic Fe in 50 
mL H 2 S0 4 (1 +9), warming if necessary to hasten reaction. Cool, 
and dil. to 1 L with H 2 0. 

3.012 Preparation of Sample 

{Caution: See 51.011, 51.025, and 51.030.) 

Use Soln /, 3.005, or if Soln I is not available, weigh samples of 
finely ground plant material (1-5 g, depending on Fe content) 
into porcelain crucibles with smooth inner surfaces, and ash 
overnight at 500-550° in furnace. Cool, add 5 mL HCI (1 +1 ), and 
heat on steam bath 15 min to dissolve Fe and to hydrolyze 
pyrophosphate. Filter into 100 mL vol. flask. Transfer insol. 
residue to filter and wash 5 times with 3 mL portions hot HCI 
(1 + 100), then with hot H 2 until washings are Cl-free. Ignite 
paper and any remaining C in Fe-free Pt crucible. Cool, add 2 
drops H 2 S0 4 and 1 mL HF, and carefully evap. to S0 3 fumes. Cool, 
add few drops HCI (1+1), and warm. Filter and wash as before 
into same vol. flask, dil. to vol., and mix. 



3.013 



Determination 



Pipet identical aliquots of Soln I, 3.005, or sample soln, 3.012, 
into 25 mL vol. flask and into test tube or small erlenmeyer. Add 
5 drops bromophenol blue indicator to aliquot in test tube, and 
titr. with 2M NaOAc soln until color matches that of equal vol. 
of pH 3.5 buffer contg same amt of indicator, Add 1 mL 
hydroquinone soln and 2 mL o-phenanthroline soln to aliquot 
in vol. flask, and adjust pH to 3.5 by adding same vol. NaOAc 
soln found necessary for aliquot in test tube. If turbidity develops 
upon adjusting pH of aliquot in test tube, add 1 mL NH 4 citrate 
soln to vol. flask before adding the NaOAc soln. Dil. to vol., mix, 
and let stand 1 hr for complete color development, and measure 
A at max., ca 510 nm. 

Prep, curve relating A to mg Fe in 25 mL by treating series of 
solns contg amts of Fe that cover usable range of instrument 
exactly as described for unknowns, detg their respective read- 
ings at max. A, ca 510 nm, and plotting these against corre- 
sponding concns of Fe. H 2 may be used as ref., and blanks 
detd to correct for amt Fe in reagents used, or blank soln itself 
may be made basis of comparison. 

3.014 Titrimetric Method (5) — Official Pinal Action 

Take appropriate aliquot of Soln I or of soln prepd as in 3.012, 
and oxidize Fe by adding soln of KMn0 4 (1 + 1000) dropwise until 
very faint permanganate color persists. Add 5 mL 10% NH 4 SCN 



and titr. with dil. TiCi 3 soin until red color disappears. (To prep, 
appropriate TiCI 3 soln, boil 5-10 mL 20% TiCI 3 with 50 mL HCI 
few min, cool, and dil. to 1 L. Stdze against std Fe soln, keep in 
dark in well-filled container, and restdze each time it is used, or 
every few hr when many detns are being made. Discard when 
decomposition is indicated by loss of color and increased titer 
against std.) 

Calcium — Official Final Action 

3.015 Macro Method {6) 

Transfer aliquot of Soln I, 3.005, to 200 mL beaker, add H 2 if 
necessary to vol. of 50 mL, heat to bp, and add 10 mL satd 
(NH 4 ) 2 C 2 4 soln and drop Me red, 2.055(i). Almost neutze with 
NH 4 0H and boil until ppt is coarsely granular. Cool, add NH 4 0H 
(1 +4) until color is faint pink (pH 5.0), and let stand 5*4 hr. Filter, 
and wash with H 2 at room temp, until filtrate is oxalate-free. 
(Reserve filtrate and washings for Mg detn, 3.017.) 

Break point of filter with Pt wire, and wash ppt into beaker in 
which Ca was pptd, using stream of hot H 2 0. Add ca 10 mL 
H 2 S0 4 (1 +4), heat to ca 90°, add ca 50 mL hot H 2 0, and titr. with 
0.05/V KMn0 4 . Finally add filter paper to soln and complete titrn. 

3.016 Micro Method (7) 

Weigh 2 g sample into small crucible and ignite in furnace at 
500-550°. Dissolve ash in HCI (1+4) and transfer to 100 mL 
beaker. Add 5 mL HCI and evap. to dryness on steam bath to 
dehydrate Si0 2 . Moisten residue with 5 mL HCI, add ca 50 mL 
H 2 0, heat few min on steam bath, transfer to 100 mL vol. flask, 
cool quickly to room temp., dil. to vol., shake, and filter, 
discarding first portion of filtrate. 

Pipet 15 mL aliquot into conical-tip centrf. tube contg 2 mL 
satd (NH 4 ) 2 C 2 4 soln and 2 drops Me red, 2.0550). Add 2 mL 
HOAc (1+4), rotating tube to mix contents thoroly. Add NH 4 0H 
(1 +4), while intermittently rotating tube, until soln is faintly alk.; 
then add few drops of the HOAc until color is faint pink (pH 5.0). 
(It is important at this point to rotate tube so that last bit of liq. 
in conical tip has required color.) Let stand ^4 hr; then centrf. 
15 min. (Ppt should be in firm lump in tip of tube.) Remove 
supernate, using suction device, Fig. 3:01, taking care not to 
disturb ppt. Wash ppt by adding 2 mL NH 4 OH (1+49), rotating 
tube to break up ppt. (It may be necessary to jar tube sharply.) 
Centrf. 10 min, again remove supernate, and wash with reagent 
as before. Repeat washing of ppt 3 times. 

After removing last supernate, add 2 mL H 2 S0 4 (1 +4) to tube, 
break up ppt as before, heat on steam bath to 80-90°, and titr. 




FIG. 3: 01 —Suction device used in micro method for determining calcium 



AOAC Methods (1980) 



Sodium 



33 



in tube with 0.02/V KMn0 4 , rotating liq. during titrn to attain 
proper end point. If tube cools to <60° during titrn, as indicated 
by slow reduction of KMn0 4 , reheat in steam bath few min and 
complete titrn. Perform blank on identical vol. H 2 S0 4 in similar 
tube heated to same temp, to det. vol. KMn0 4 soln necessary 
to give end point color. Subtract this value from buret reading. 
1 mL 0.02/V KMn0 4 = 0.000400 g Ca. Report as % Ca. 

3.017 Magnesium [8) — Official Final Action 

{Caution: See 51.026.) 

To combined filtrate and washings from Ca detn, 3.015, add 
30 mL HN0 3 and evap. to dryness to decompose NH 4 salts. Take 
up with 5 mL HCI and dil. to ca 100 mL with H 2 0. Add 5 mL 10% 
Na citrate soin and 10 mL 70% {NH 4 ) 2 HP0 4 soln, or enough to 
ppt all the Mg. Add NH 4 OH (1+4) with const stirring (using 
policeman) until soln is faintly alk. and ppt forms; then add 25 
mL NH 4 OH, stir vigorously until ppt is granular, and keep in cool 
place overnight. Filter, and wash Cl-free with cold NH 4 OH (1+10). 
Incinerate in furnace at 500-550° until all C is oxidized, then at 
900-950° ca 4 hr to form Mg 2 P 2 7 . Cool, and weigh as Mg 2 P 2 7 . 
(If sample is excessively high in Mn, dissolve residue in HN0 3 , 
det. Mn as in 3.018, and correct Mg 2 P 2 7 for Mn 2 P 2 7 .) Report 
as % Mg. 

3.018 Manganese (9) — Official Final Action 

To aliquot of Soln /, 3.005, contg 0.2-0.5 g ash, add 15 mL 
H 2 S0 4 and evap. to ca 30 mL. Add 5-10 mL HN0 3 and continue 
evapn. (Do not evap. until dense fumes appear, because Fe 2 (S0 4 ) 3 
then dissolves with difficulty. HN0 3 may be present, but not 
HCI.) Add H 2 0, little at time, heat until Fe salts dissolve, and dil. 
to ca 150 mL. Add 0.3 g KIO A , or its equiv. in HI0 4 , in small 
portions, boil few min or until color of KMn0 4 shows no further 
increase in intensity, and let cool. 

Prep, std as follows: To vol. H 2 equal to sample add 15 mL 
H 2 S0 4 and enough pure Fe(N0 3 ) 3 , free from Mn, to equal approx. 
amt of Fe in sample. Add measured vol. 0.1/V KMn0 4 until color 
is slightly darker than sample, then add 0.3 g KI0 4 , and boil few 
min. When cool, transfer sample and std to 250 mL vol. flasks 
and dil. to vol. with H 2 0. (If color is weak, it may be necessary 
to dil. to <250 mL.) Measure A with photometer or spectrophtr 
set at max., ca 530 nm. Report as % Mn. 

3.019 it Potassium and Sodium * 

Official Final Action 

Ignition, removal of Fe, Al, and P with NH 4 OH, sulfate as BaS0 4 , 
Ca as oxalate, NH 3 by ignition, and final weighing as NaCI + 
KCI. See 3.015, 11th ed. 

Potassium and/or Sodium 

Flame Photometric Method {10) 
Official Final Action 



3.020 



Reagents 

-1000 ppm K. Dissolve 1.907 g dry 



(a) Potassium stock soln.- 
KCI in H 2 and dil. to 1 L 

(b) Sodium stock soln.— -1000 ppm Na. Dissolve 2.542 g dry 
NaCI in H 2 and dil. to 1 L 

(c) Lithium stock soln. — 1000 ppm Li. Dissolve 6.108 g LiCI in 
H 2 and dil. to 1 L. (Needed only if internal std method of 
evaluation is to be used.) 

(d) Ammonium oxalate stock soln. — 0.24/V. Dissolve 17.0 g 
(NH 4 ) 2 C 2 4 .H 2 in H 2 and dil. to 1 L 

* Surplus method— see inside front cover. 



(e) Extracting solns. — (7) For potassium. — For internal std 
method, dil. required vol. LiCI stock soln to 1 L; otherwise use 
H 2 0. (2) For sodium. — To 250 mL NH 4 oxalate stock soln add 
required vol. LiCI stock soln (if internal std method is used) and 
dil. to 1 L. If internal std requirements are same for both Na and 
K detns, this reagent may be used as common extg soln. 

3.021 Preparation of Standard Solutions 

Dil. appropriate aliquots of stock solns to prep, series of stds 
contg K and/or Na in stepped amts (including 0) to cover 
instrument range, and Li and NH 4 oxalate (if required) in same 
concns as in corresponding extg solns. (If common extg soln is 
used, 1 set of stds contg both K and Na suffices.) 

3.022 Sample Extraction 

Transfer weighed portion of finely ground and well mixed 
sample to erlenmeyer of at least twice capacity of vol. of extg 
soln to be used. Add measured vol. extg soln, stopper flask, and 
shake vigorously at frequent intervals during s*15 min. Filter 
thru dry, fast paper. If paper clogs, pour contents onto addnl 
fresh paper and combine filtrates. Use filtrate for detn. 

Note: Do not make exts more coned than required for instru- 
ment because there is tendency toward incomplete extn as ratio 
of sample wt to vol. extg soln increases. Prep. sep. exts for K 
and Na when their concns in sample differ greatly. For K, use 
wt sample =£0.1 g/50 mL extg soln; for low Na concns use sM.O 
g/50 mL extg soln; and for higher concns, prep, weaker exts by 
reducing ratio of sample to extg soln rather than by dilg stronger 
exts. 



3.023 



Determination 



{Caution: See 51.007.) 



Rinse all glassware used in Na detn with dil. HN0 3 , followed 
by several portions H 2 0. Protect solns from air-borne Na con- 
tamination. 

Operate instrument according to manufacturer's instructions. 
Permit instrument to reach operating equilibrium before use. 
Aspirate portions of std solns toward end of warm-up period 
until reproducible readings for series are obtained. 

Run stds, covering concn range of samples involved, at 
frequent intervals within series of sample soln detns. Repeat 
this operation with both std and sample solns enough times to 
result in reliable av. reading for each soln. Plot curves from 
readings of stds, and calc. % K and/or Na in samples. 

* Potassium — Official Final Action * 

3.024 Platinic Chloride Method 
See 3.020, 11th ed. 

3.025 Perchloric Acid Method {11) 
See 3.021, 11th ed. 

3.026 Rapid Method for Potassium Only 
See 3.022, 11th ed. 

Sodium Only 

Uranyl Acetate Method {12) — Official Final Action 

3.027 Reagent 

Magnesium uranyl acetate soin: 

(a) Uranyl acetate soln.— To 85 g U0 2 (OAc} 2 .2H 2 in 1 L vol. 
flask add 60 g HOAc and H 2 to ca 900 mL Heat to dissolve, 
cool, and dil. to vol. with H 2 0. {Caution: See 51.083.) 



34 



3. Plants 



AOAC Methods (1980) 



(b) Magnesium acetate soln. — To 500 g Mg(OAc) 2 .4H 2 in 1 
L vol. flask add 60 g HOAc and H 2 to ca 900 ml Heat to 
dissolve, cool, and dil. to vol. with H 2 0. 

Reheat (a) and (b) sep. to ca 70° until all salts dissolve. Mix 
the solns at this temp, and let cool to ca 30°. Place vessel contg 
mixed reagent in H 2 bath at 20°, and hold 1-2 hr at 20°, or until 
slight excess of salts has crystd out. Filter thru dry filter into dry 
bottle. 



3.028 



Determination 



Moisten 1-10 g sample with H 2 S0 4 (1 + 10), dry in oven, and 
ignite in furnace at 500-550° to destroy org. matter. Heat residue 
on steam bath with 2-5 ml_ HCI, add ca 40 mL H 2 0, and heat to 
bp. Add enough 5% CaCI 2 soln to ensure pptn of all phosphates. 
Ppt phosphates by making slightly alk. with NH 4 OH. Filter, and 
evap. to =£5 mL if no salts sep. Cool, add 100 mL Mg uranyl 
acetate soln, place mixt. in H 2 bath at 20°, and either stir 
vigorously 45 min or let stand 24 hr at this temp. Filter with 
suction, and wash with alcohol satd with Na-Mg-uranyl acetate. 
Dry 30 min at 105-110°, cool, and weigh. Wt Na-Mg-uranyl 
acetate x 0.0153 =wt Na. 

Cobalt — Official Final Action 
{Caution: See 51.011, 51.040, 51.049, and 51.068.) 



Nitrosocresoi Method (13) 



3.029 



Reagents 



(Make all distns in Pyrex stills with f joints. 
Store reagents in g-s Pyrex bottles.) 



(a) Redistilled water. — Distil twice, or pass thru column of ion 
exchange resin (IR-100A, H-form, or equiv.) to remove heavy 
metals. 

(b) Hydrofluoric acid. — 48%. Procurement in vinyl plastic 
bottles is advantageous. 

(c) Perchloric acid. — 60%. No further purification necessary. 

(d) Hydrochloric acid.— -(1 + 1). Add equal vol. HCI to distd H 2 
and distil. 

(e) Ammonium hydroxide. — (1 + 1). Distil coned NH 4 OH into 
equal vol. redistd H 2 0. 

(f) Ammonium hydroxide.— 0.02/V. Add 7 mL of the NH 4 OH 
(1 + 1) to 2.5 L redistd H 2 0. 

(g) Carbon tetrachloride. — Distil over CaO, passing distillate 
thru dry, acid-washed filter paper. Used CCI 4 may be recovered 
as in 3.044(a). 

(h) Dithizone — Dissolve 0.5 g dithizone in 600-700 mL CCI 4 
(tech. grade is satisfactory). Filter into 5 L separator contg 2.5- 
3.0 L 0.02/V NH 4 0H, shake well, and discard CCI 4 layer. Shake 
with 50 mL portions redistd CCI 4 until CCI 4 phase as it seps is 
pure green. Add 1 L redistd CCI 4 and acidify slightly with the HCI 
(1 +1 ). Shake the dithizone into CCI 4 layer and discard aq. layer. 
Store in cool, dark place, preferably in refrigerator. 

(i) Ammonium citrate soln. — 40%. Dissolve 800 g citric acid 
in 600 mL distd H 2 0, and, while stirring, slowly add 900 mL 
NH 4 OH. Reaction is exothermic; take care to prevent spattering. 
Adjust pH to 8.5, if necessary. Dil. to 2 L and ext with 25 mL 
portions dithizone soln until aq. phase stays orange and CCI 4 
remains predominantly green. Then ext soln with CCI 4 until all 
orange is removed. 

(j) Hydrochloric acid.—OAN. Dil. 16.6 mL of the HCI (1+1) to 
1 L with redistd H 2 0. 

(k) Hydrochloric acid.—Q.QIN. Dil. 100 mL of the 0.1/V HCI to 
1 L with redistd H 2 0. 

(I) Sodium hydroxide soln.— IN. Dissolve 40 g NaOH in 1 L 
redistd H 2 0. 



(m) Borate buffer.— pH 7.8. Dissolve 20 g H 3 B0 3 in 1 L redistd 
H 2 0. Add 50 mL 1/V NaOH and adjust pH, if necessary. Equal 
vols borate buffer and 0.01/V HCI should give soln of pH 7.9. 

(n) Borate buffer.— -pH 9.1. To 1 L borate buffer, pH 7.8, add 
120 mL 1/V NaOH and adjust pH, if necessary. 

(o) Skellysoive B. — Essentially n-hexane. Purify by adding 
20-30 g silica gel/L, let stand several days, and distil. Available 
from Getty Refining and Marketing Co., PO Box 1650, Tulsa, OK 
74102. 

(p) Cupric acetate soln. — Dissolve 10 g Cu(OAc) 2 .H 2 in 1 L 
redistd H 2 0. 

(q) o-Nitrosocresoi soln.— Dissolve 8.4 g anhyd. CuCI 2 and 8.4 
g NH 2 OH.HCI in 900 mL H 2 0. Add 8 mL m-cresol (Eastman Ko- 
dak Co., practical grade) and stir vigorously while slowly adding 
24 mL 30% H 2 2 . Stir mech. 2 hr at room temp. (Standing for 
longer periods results in excessive decomposition.) Add 25 mL 
HCI and ext o-nitrosocresol with four 1 50 mL portions Skellysoive 
B, (o), in large separator. Then add addnl 25 mL HCI and again 
ext with four 150 mL portions Skellysoive B. Wash combined 
Skellysoive B exts twice with 50-100 mL portions 0.1/V HCI and 
twice with 50-100 mL portions redistd H 2 0. Shake o-nitrosocre- 
sol soln with successive 50-100 mL portions 1% Cu(OAc) 2 soln 
until aq. phase is no longer deep blood-red. When light purple 
is evident, extn is complete. Discard Skellysoive B phase, acidify 
aq. soln of Cu salt with 25 mL HCI, and ext reagent with two 500 
mL portions Skellysoive B; wash twice with 1 50-200 mL portions 
0.1/V HCI and several times with 150-200 mL portions redistd 
H 2 0. Store o-nitrosocresol soln in refrigerator at ca 4°. Reagent 
is stable 5=6 months. 

(r) Sodium o-nitrosocresol soln. — Ext 100 mL o-nitrosocresol 
by shaking with two 50 mL portions borate buffer, pH 9.1, in 
separator. (If this is carried out as 2 extns, resulting reagent is 
more coned. It is important that total vol. o-nitrosocresol soln 
equal total vol. buffer.) 

(s) Cobalt std solns.— {1) Stock soln.— Heat CoS0 4 .7H 2 in 
oven at 250-300° to const wt (6-8 hr). Weigh exactly 0.263 g of 
the CoS0 4 and dissolve in 50 mL redistd H 2 and 1 mL H 2 S0 4 . 
Dil. to 1 L. {2) Working so//?. —0.5 ^g/mL. Transfer 5 mL stock 
soln to 1 L vol. flask and dil. to vol. with redistd H 2 0. 

(t) Hydroxyiamine acetate buffer. — pH 5.1 ±0.1. Dissolve 10 
g NH 2 OH.HCI and 9.5 g anhyd. NaOAc in 500 mL redistd H 2 0. 



3.030 



Apparatus 



(a) Platinum dishes.— Approx. 70mL;forashing. 

(b) Automatic dispensing burets. — 100 mL; type that can be 
fitted to ordinary 5 lb reagent bottle and filled by means of 
aspirator bulb is most convenient. 

(c) Wooden separator rack. — Twelve- unit 125 mL separator 
size is convenient for dithizone extns. Rack is fitted across top 
with removable bar padded with sponge rubber so all 12 
separators can be shaken as unit. 

(d) Racks.— Consisting of 5 x 5 x 65 cm (2 x 2 x 25") wooden 
bars with holes drilled at close intervals to take 50 mL centrf. 
tubes fitted with No. 13 f glass stoppers. To make these tubes, 
ream out necks of heavy-wall Pyrex centrf. tubes (Rockefeller 
Institute type) with f C rod and grind to take f stopper. Place 
tubes upright in one section, and place other section (fitted with 
sponge rubber disks 13 mm thick in bottom of holes) across 
their tops. Fasten 2 sections at ends with removable rubber 
connectors made from ordinary tubing of convenient size, so 
that any number of tubes can be shaken as unit. Use these tubes 
for reaction of Co with nitrosocresoi, extn of complex into 
Skellysoive B, and washing of Skellysoive B soln. 

(e) Shaking machine.— Mech. shaker giving longitudinal 
stroke of 5 cm at ca 180 strokes/min; use to make dithizone 
extns and to ext Co complex, or shake by hand. 



AOAC Methods (1980) 



Cobalt 



35 



3.031 



Cleaning of Glassware 



Clean 120 mi_ Pyrex separators for dithizone extns by initially 
soaking 30 min in hot HN0 3 and rinsing several times with H 2 0. 
As added precaution, shake with several portions dithizone in 
CCI 4 . After use, clean by rinsing with H 2 0, drain, and stopper to 
avoid contamination. It is not necessary to clean every time with 
acid. Repeat HN0 3 cleaning if blanks are unusually high. 

Clean 50 ml_ g-s Pyrex centrf. tubes by soaking 30 min in 
HN0 3 followed by several rinsings in H 2 0. 

Completely submerge pipets in cylinder of chromic acid 
cleaning soln overnight, rinse several times with H 2 0, and 
suspend upright in rack to dry. 

Wash all other glassware thoroly in detergent and rinse well 
with tap H 2 followed by dip in chromic acid cleaning soln. 
Rinse off cleaning soln with tap H 2 followed by several distd 
H 2 rinses. 

Clean Pt by scrubbing with sea sand followed by boiling in 
HCI (1+2) 30 min, and rinse several times with H 2 0. 

3.032 Preparation of Sample 

See 3.002(a). Oven-dry all plant material 48 hr and prep, for 
ashing by either of following methods: 

(a) Grind material in Wiley mill equipped with stainless steel 
sieve, mix thoroly by rolling, and sample by quartering. 

(b) Using stainless steel shears, cut material by hand fine 
enough for convenient sampling. 

3.033 Ashing of Samples 

{Caution: See 51.011, 51.025, and 51.028.) 

Weigh 6 g dry plant tissue into clean Pt dish. Cover with Pyrex 
watch glass and place in cool furnace; heat slowly to 500° and 
hold at this temp, overnight. Remove sample and cool. Wet 
down ash carefully with fine stream redistd H 2 0. From dis- 
pensing buret, slowly add 2-5 mL HCI0 4 , dropwise at first to 
prevent spattering. Add ca 5 mL HF, evap. on steam bath, 
transfer to sand bath, and keep at medium heat until fuming 
ceases. 

Cover with Pyrex watch glass, return to partially cooled 
furnace, heat gradually to 600°, and keep at this temp. 1 hr. 
Remove sample and cool. Add 5 mL HCI (1 + 1) and ca 10 mL 
redistd H 2 0. Replace cover glass and warm on steam bath to 
dissolve. (Usually clear soln essentially free of insol. material is 
obtained.) Transfer sample to 50 mL vol. flask, washing dish 
several times with redistd H 2 0, dil. to vol., and mix thoroly. (Pt 
dishes can ordinarily be used several times between sand and 
acid cleanings.) 

3.034 Dithizone Extraction 

[Caution: See 51.011(b), 51.028(a) and (d), and 51.049.} 

Transfer suitable aliquot (2-3 g dry material) to 120 mL 
separator (use petroleum jelly as stopcock lubricant). Add 5 mL 
NH 4 citrate soln and 1 drop phthln; adjust to pH 8.5 with NH 4 OH 
(1+1). If ppt forms, add addnl NH 4 citrate. Add 10 mL dithizone 
in CCU and shake 5 min. Drain CCI 4 phase into 100 mL beaker. 
Repeat as many times as necessary, using 5 mL dithizone soln 
and shaking 5 min each time. Extn is complete when aq. phase 
remains orange and CCI 4 phase remains predominantly green. 
Then add 10 mL CCI 4 , shake 5 min, and combine with CCI 4 ext. 
Final 10 mL CCI 4 should be pure green. If not, extn was 
incomplete and must be repeated. 

Add 2 mL HCI0 4 to combined CCI 4 exts, cover beaker with 
Pyrex watch glass, and digest on hot plate until colorless. 
Remove cover glass and evap. slowly to dryness. (If sample is 
heated any length of time at high temp, when dry, losses of Co 



may occur. Heat only enough to evap. completely to dryness. 
If free acid remains, it interferes with next step where pH control 
is important.) 

Add 5 mL 0.01/V HCI to residue. Heat slightly to assure soln. 
If Cu is to be detd, transfer with redistd H 2 to 25 mL vol. flask, 
and dil. to vol. Transfer 20 mL aliquot to 50 mL g-s centrf. tube 
or 60 mL separator and reserve remainder for Cu detn, 3.043. 
If Cu is not to be detd, transfer entire acid soln with redistd H 2 
to centrf. tube or separator. 



3.035 



Determination 



Add 5 mL borate buffer, pH 7.8, and 2 mL freshly prepd Na 
o-nitrosocresol soln to sample soln. Add exactly 5 mL Skellysolve 
B and shake 10 min. Remove aq. phase by moderate suction 
thru finely-drawn glass tube. To Skellysolve B layer add 5 mL 
Cu(OAc) 2 soln and shake 1 min to remove excess reagent. Again 
remove and discard aq. phase. Wash Skellysolve B by shaking 
1 min with 5 mL redistd H 2 0, removing aq. layer as before; 
finally shake Skellysolve B 1 min with 5 mL NH 2 OH-NaOAc buffer 
to reduce Fe. Transfer Skellysolve B soln of the Co complex to 
5 cm cell and read in spectrophtr as close as possible to point 
of max. A, 360 nm. 

3.036 Blanks and Standards 

With each set of detns include ashing blank and Co stds of 
0.0, 0.5, and 1.0 ^g. Beer's law holds for this range. A of 0.0 fig 
point should be <0.05. If above, repurify o-nitrosocresol by 
transferring alternately to aq. phase as Cu salt and to Skellysolve 
B phase as free compd after acidifying aq. phase. 

It is also advisable to include std sample with each set of 
samples to detect contamination or unusual losses of Co in 
method. Com. buckwheat flour contg 0.05 ppm Co has proved 
satisfactory for this purpose. 

3.037 Calculations 

Express results in terms of ppm Co, based upon dry wt of 
sample, 
ppm Co = (fig Co/mL dithizone aliquot) 

x (mL total soln/g dry sample) 

Value for fig Co is obtained from curve minus ashing blank. 



Nitroso-R-Salt Method {14) 



3.038 



Reagents 



Those listed in 3.029 and following: 

(a) Nitroso~R-salt soln. — 0.2%. Dissolve 2 g powd nitroso-R- 
salt (Eastman Kodak Co., No. 1124) in redistd H 2 0, 3.029(a), and 
dil. to 1 L. 

(b) Dilute nitric acid.— (1+1). Dil. HN0 3 with equal vol. H 2 
and redistil in Pyrex a pp. Store in Pyrex bottles. 

(c) Bromine water. — Satd soln of Br in redistd H 2 0, 3.029(a). 

(d) Citric acid soln. — 0.2/V. Use special reagent grade Pb-free 
citric acid. 



3.039 Preparation and Ashing of Samples 

Proceed as in nitrosocresol method, 3.032-3.033, thru "(Usu- 
ally clear soln essentially free of insol. material is obtained.)" 
except use 10 g instead of 6 g dry plant tissue. 



3.040 



Dithizone Extraction 



Transfer entire soln to 120 mL separator, and proceed as in 
3.034, thru "If free acid remains . . . pH control is important.)" 
Dissolve in 1 mL citric acid soln, (d), transfer to 25 mL vol. flask, 
and dil. to vol. with redistd H 2 0, 3.029(a). 



36 



3. Plants 



AOAC Methods (1980) 



3.041 



Determination 



Transfer suitable aliquot (ca 8 g dry material) of citric acid 
soln, 3.040, to 50 mL beaker. Evap. to 1-2 mL. Add 3 mL borate 
buffer, 3.029(n), and adjust pH to 8.0-8.5 with NaOH (check 
externally with phenol red). (Vol. ^5 mL) Add 1 mL nitroso-R- 
salt soln slowly with mixing. Boil 1-2 min and add 2 mL dil. 
HN0 3 . Boil 1-2 min, add 0.5-1.0 mL Br-H 2 0, cover with watch 
glass, and let stand warm 5 min. Boil 2-3 min to remove excess 
Br (use effective fume removal device). Cool, and dil. to 10 or 
25 mL (depending on length of light path in absorption cell). 
Transfer to cell and read at 500 nm within 1 hr. Prep, stds contg 
0.5, 1, 2, 3, and 4 /xg Co and add 1 mL citric acid soln, 3.038(d), 
to each. Proceed as for unknowns, beginning "Evap. to 1-2 mL." 



Copper [14) — Official Final Action 



3.042 



Reagents 



Those listed in 3.029 and following: 

(a) Sodium diethyidithiocarbamate soln. — 0.1 %. Freshly 
prepd in redistd H 2 0, 3.029(a). 

(b) Copper std soln.— 1 ^g/mL Dissolve 0.3929 g CuS0 4 .5H 2 
in redistd H 2 0, 3.029(a), add 5 mL H 2 S0 4 , dil. to 1 L, and mix. 
Take 10 mL aliquot, add 5 mL H 2 S0 4 , dil to 1 L, and mix. 



3.043 



Determination 



Transfer aliquot (0.5-1 g dry material) from soln obtained 
from 3.034 or 3.040 to 125 mL separator. Add 2 mL NH 4 citrate 
soln, 1 drop phthln, 5 mL Na diethyidithiocarbamate soln, and 
NH 4 OH (1 + 1), 3.029(e), until pink. Add 10 mL CCI 4 and shake 5 
min. Drain CCI 4 , centrf. 5 min, transfer to absorption cell, and 
read at max. A, ca 430 nm, 

Prep, std curve with 0, 1, 5, 10, 15, and 20 /xg Cu treated as 
above. 

Zinc — Official Final Action 



Mixed Color Method {15) 



3.044 



Reagents 

(Redistil all H 2 Ofrom Pyrex. Treat all glassware with HN0 3 (1 + 1) 

or fresh chromic acid cleaning soln. Rinse repeatedly with 

ordinary distd H 2 and finally with Zn-free H 2 0.) 

(a) Carbon tetrachloride.— Use ACS grade without purifica- 
tion. If tech. grade is used, dry with anhyd. CaCI 2 and redistil in 
presence of small amt CaO. (Used CCI 4 may be reclaimed by 
distn in presence of NaOH (1 + 100) contg small amts of Na 2 S 2 3 , 
followed by drying with anhyd. CaCI 2 and fractional distn in 
presence of small amts of CaO.) (Caution: See 51.011(b) and 
51.049.) 

(b) Zinc std solns.— U) Stock sofn.-l mg/mL Place 0.25 g 
pure Zn in 250 mL vol. flask. Add ca 50 mL H 2 and 1 mL H 2 S0 4 ; 
heat on steam bath until all Zn dissolves. Dil. to vol. and store 
in Pyrex vessel. (2) Working soln.— 10 /^g/mL. Dil. 10 mL stock 
soln to 1 L. Store in Pyrex vessel. 

(c) Ammonium hydroxide soln. — 1/V. With all-Pyrex app. distil 
NH 4 OH into H 2 0, stopping distn when half has distd. Dil. distillate 
to proper concn. Store in g-s Pyrex vessel. 

(d) Hydrochloric acid.— 1/V, Displace HCI gas from HCI in glass 
flask by slowly adding equal vol. H 2 S0 4 from dropping funnel 
that extends below surface of the HCI. Conduct displaced HCI 
gas thru delivery tube to surface of H 2 in receiving flask (no 
heat is necessary). Dil. to proper concn. Use of 150 mL each of 
HCI and H 2 S0 4 will yield 1 L purified HCI soln of concn >1/V. 

(e) Diphenylthiocarbazone (dithizone) soln. — Dissolve 0.20 g 
dithizone in 500 mL CCI 4 , and filter to remove insol. matter. Place 
soln in g-s bottle or large separator, add 2 L 0.02/V NH 4 OH (40 



mL 1/V NH 4 OH dild to 2 L), and shake to ext dithizone into aq. 
phase. Sep. phases, discard CCI 4 , and ext ammoniacal soln of 
dithizone with 100 mL portions CCI 4 until CCI 4 ext is pure green. 
Discard CCl 4 after each extn. Add 500 mL CCI 4 and 45 mL 1/V 
HCI, and shake to ext dithizone into CCI 4 . Sep. phases and discard 
aq. phase. Dil. CCI 4 soln of dithizone to 2 L with CCI 4 . Store in 
brown bottle in dark, cool place. 

(f) Ammonium citrate soln. — 0.5/W. Dissolve 226 g 
(NH 4 ) 2 HC 6 H 5 7 in 2 L H 2 0. Add NH 4 OH (80-85 mL) to pH of 
8.5-8.7. Add excess dithizone soln (aq. phase is orange-yellow 
after phases have been shaken and sepd), and ext with 100 mL 
portions CCI 4 until ext is full green. Add more dithizone if 
necessary. Sep. aq. phase from CCI 4 and store in Pyrex vessel. 

(g) Carbamate soln. — Dissolve 0.25 g Na diethyidithiocarba- 
mate in H 2 and dil. to 100 mL with H 2 0. Store in refrigerator in 
Pyrex bottle. Prep, fresh after 2 weeks. 

(h) Dilute hydrochloric acid.— 0.02/V. Dil. 100 mL 1/V HCI to 
5L. 

3.045 Preparation of Solutions 

To reduce measuring out reagents and minimize errors due 
to variations in composition, prep. 3 solns in appropriate amts 
from reagents and store in Pyrex vessels, taking care to avoid 
loss of NH 3 from Solns 1 and 2. Discard solns after 6-8 weeks 
because Zn increases slowly with storage. Det. std curve for 
each new set of reagents. Following amts of Solns 1 and 2 and 
2 L dithizone soln are enough for 100 detns: 

(1) Soln /.—Dil. 1 L 0.5/W NH 4 citrate and 140 mL 1/V NH 4 OH 
to4L. 

(2) Soln 2.— Dil. 1 L 0.5M NH 4 citrate and 300 mL 1/V NH 4 OH 
to 4.5 L. Just before using, add 1 vol. carbamate soln to 9 vols 
NH 3 -NH 4 citrate soln to obtain vol. of Soln 2 immediately required. 

Note: If Zn-free reagents have been prepd, they can be used 
to test chemicals for Zn. Certain lots of NH 4 OH and HCI are 
sufficiently free of Zn to be used without purification. 



3.046 



Ashing 



Ash 5 g finely ground, air-dried plant material in Pt dish in 
furnace at 500-550°. Include blank detn. Moisten ash with little 
H 2 0; then add 10 mL 1/V HCI (more if necessary) and heat on 
steam bath until all substances sol. in HCI are dissolved. Add 
5-1 mL hot H 2 0. Filter off insol. matter on 7 cm paper (Whatman 
No. 42, or equiv., previously washed with two 5 mL portions hot 
1/V HCI, then washed with hot H 2 until HCI-free), and collect 
filtrate in 100 mL vol. flask. Wash filter with hot H 2 until 
washings are not acid to Me red. Add 1 drop Me red, 2.055(i), 
to filtrate in 100 mL flask; neutze with W NH 4 OH and add 4 mL 
1/V HCI. Cool, and dil. to vol. with H 2 0. 



3.047 



First Extraction 



(Sepn of dithizone complex-forming 
metals from ash soln) 



Pipet aliquot of ash soln contg ^30 fig Zn into 125 mL Squibb 
separator. Add 1 mL 0.2/V HCI for each 5 mL ash soln <10 mL 
taken, or 1 mL 0.2/V NH 4 OH for each 5 mL >10 mL taken. (10 mL 
aliquot is usually satisfactory in analysis of plant materials.) Add 
40 mL Soln 1 and 10 mL dithizone reagent. Shake vigorously 30 
sec to ext from aq. phase the Zn and other dithizone complex- 
forming metals that may be present; then let layers sep. At this 
point excess dithizone (indicated by orange or yellow-orange 
aq. phase) must be present. If excess dithizone is not present, 
add more reagent until, after shaking, excess is indicated. Shake 
down the drop of CCI 4 ext from surface, and drain CCI 4 ext into 
second separator as completely as possible without letting any 
aq. layer enter stopcock bore. Rinse down CCI 4 ext from surface 



AOAC Methods (1980) 



Molybdenum 



37 



of aq. layer with 1-2 mL clear CCI 4 ; then drain this CCI 4 into 
second separator without letting aq. phase enter stopcock bore. 
Repeat rinsing process as often as necessary to flush ext 
completely into second separator. Add 5 mL clear CCI 4 to first 
separator, shake 30 sec, and let layers sep. (CCI 4 layer at this 
point will appear clear green if metals that form dithizone 
complexes have been completely extd from aq. phase by 
previous extn.) Drain CCI 4 layer into second separator and flush 
ext down from surface and out of separator as directed previ- 
ously. If last ext does not possess distinct clear color, repeat 
extn with 5 mL clear CCI 4 and flushing-out process until complete 
extn of dithizone complex-forming metals is assured; then 
discard aq. phase. 



3.048 



Second Extraction 



(Sepn of Cu by extn of Zn into 0.02/V HCI) 

Pipet 50 mL 0.02/V HCI into separator contg CCI 4 soln of metal 
dithizonates. Shake vigorously 1.5 min, and let layers sep. Shake 
down drop from surface of aq. phase, and as completely as 
possible drain CCI 4 phase contg all Cu as dithizonate, without 
letting any aq. phase, which contains all the Zn, enter stopcock 
bore. Rinse down CCI 4 ext from surface of aq. phase, and rinse 
out stopcock bore with 1-2 mL portions clear CCI 4 (same as in 
first extn) until all traces of green dithizone have been washed 
out of separator. Shake down drop of CCI 4 from surface of aq. 
phase, and drain CCI 4 as completely as possible without letting 
any aq. phase enter stopcock bore. Remove stopper from 
separator and lay it across neck until small amt of CCI 4 on 
surface of aq. phase evaps. 



3.049 



Final Extraction 



(Extn of Zn in presence of carbamate reagent) 

Pipet B0 mL Soln 2 and 10 mL dithizone soln into 50 mL 0.02/V 
HCI soln contg the Zn. Shake 1 min and let phases sep. Flush out 
stopcock and stem of separator with ca 1 mL CCI 4 ext; then 
collect remainder in test tube. Pipet 5 mL ext into 25 mL vol. 
flask, dil. to vol. with clear CCI 4 , and measured with spectrophtr 
set at absorption max., ca 525 nm. {Caution: Protect final ext 
from sunlight as much as possible and read within 2 hr.) 

Det. Zn present in aliquot from curve relating A and concn, 
correct for Zn in blank, and calc. % Zn in sample. 



3.050 



Standard Curve 



Place 0, 5, 10, 15, 20, 25, 30, and 35 mL Zn working std soln 
in 100 mL vol. flasks. To each flask add 1 drop Me red and 
neutze with 1/V NH 4 OH; then add 4 mL 1/V HCI and dil. to vol. 
Proceed exactly as for ash solns, beginning with first extn, and 
using 10 mL aliquots of each oftheZn solns(0, 5, 10, 15, 20, 25, 
30, and 35 fig Zn, resp.). Construct std curve by plotting /xg Zn 
against A. 



Sing/e Color Method (76) 



3.051 



Reagents 

See 3.044^3.045 plus following: 

(a) Dilute dithizone sofn.—DW. 1 vol. dithizone soln, 3.044(e), 
with 4 vols CCI 4 . 

(b) Carbamate soin. — Dissolve 1.25 g Na diethyldithiocarba- 
mate in H 2 and dil. to 1 L. Store in refrigerator and prep, fresh 
after long periods of storage. 

(c) Dilute ammonium hydroxide.— Dil. 20 mL 1/V NH 4 OH, 
3.044(c), to 2 L 



3.052 Ashing 

Weigh 2 g sample finely ground plant material into well- 
glazed porcelain, Vycor, or Pt crucible, include crucible for blank 
detn, and heat in furnace at 500-550° until ashing is complete. 
Cool, moisten ash with little H 2 0, add 10 mL 1/V HCI (more if 
necessary to ensure excess of acid), and heat on steam bath 
until all sol. material dissolves. Add few mL hot H 2 and filter 
thru quant, paper into 200 mL vol. flask. Wash paper with hot 
H 2 until washings are not acid to Me red. Add 2 drops Me red 
soln to filtrate, neutze with 1/V NH 4 OH, add exactly 3.2 mL 1/V 
HCI, dil. to vol. with H 2 0, and mix. 



3.053 



Formation of Zinc Dithizonate 



(Removal of interferences and 
sepn of excess dithizone) 



Pipet aliquot of ash soln contg =^15/xg Zn into 125 mL amber 
glass separator. (25 mL aliquot is usually satisfactory.) If nec- 
essary to use different vol., add 0.4 mL 0.2/V HCI for each 5 mL 
less, or 0.4 mL 0.2/V NH 4 OH for each 5 mL more, than 25 mL 
taken. If <25 mL of the soln is taken, add H 2 to 25 mL. 

Add 10 mL dithizone reagent, 3.044(e), to aliquot in separator 
and shake vigorously 1 min. Let layers sep. and discard CCI 4 
layer. Add 2 mL CCI 4 to aq. soln, let layers sep., and discard 
CCI 4 . Repeat this rinsing once. Then add 5 mL CCI 4 , shake 
vigorously 15 sec, let layers sep., and discard CCI 4 . Rinse once 
more with 2 mL CCI 4 as above. Discard CCI 4 layer and let CCI 4 
remaining on surface of soln in funnel evap. before proceeding. 

Add 40 mL NH 4 citrate Soln 1, 3.045(1), 5 mL carbamate soln, 
3.051(b), and 25 mL dil. dithizone reagent, 3.051(a). Accurately 
add carbamate and dithizone reagents from pipet or buret Shake 
vigorously 1 min. Let layers sep. and draw off aq. layer thru fine 
tip glass tube connected to aspirator with rubber tubing. To 
remove excess dithizone from CCI 4 layer, add 50 mL 0.01/V 
NH 4 OH and shake vigorously 30 sec. 



3.054 



Determination 



Dry funnel stem with pipestem cleaner and flush out with ca 
2 mL of the Zn dithizonate soln. Collect adequate portion of 
remaining soln in 25 mL erlenmeyer, or other suitable container, 
and stopper tightly. (Amber glass containers are convenient, but 
colorless glassware will suffice if solns are kept in dark until A 
readings are made.) 

Measure A of each soln against CCI 4 with spectrophtr set at 
absorption max., ca 535 nm. Correct for Zn in blank detns. Calc. 
amt Zn present in soln from curve relating concn and/4. 



3.055 



Standard Curve 



Into 200 mL vol. flasks place 0, 2, 4, 6, 8, 10, 12, and 14 mL, 
resp., Zn working std soln. To each flask add 2 drops Me red 
soln, neutze with 1/V NH 4 OH, add 3.2 mL 1/V HCI, and dil. to vol. 
with H 2 0. Pipet 25 mL aliquots of each of these solns, contg 0, 
2.5, 5, 7.5, 10, 12.5, 15, and 17.5 fxg Zn, resp., into amber glass 
separators, and proceed as for ash solns, 3.053, beginning with 
second par. Det. A of each soln and plot values against corre- 
sponding amts Zn. 



Molybdenum (77)— Official Final Action 

3.056 Apparatus 

Photoelectric colorimeter or spectrophotometer. — Capable of 
isolating band at ca 465 nm. (Photometer equipped with filter 
with max. T at 440-460 nm and 1 cm cells of 10 mL capacity is 
suitable.) 



38 



3. Plants 



AOAC Methods (1980) 



3.057 



Reagents 



(a) Isoamyi alcohol. — Reagent grade 3-methyl-1-butanol, bp 
128-132°. 

(b) Dilute hydrochloric acid.—{1) 20% so/A7. — Dil. coned HCI 
to ca 20% HCI (1 + 1.85). {2) 6N soln.— Stdze to second decimal 
place. 

(c) Iron std soln.— 100 /xg/mL Dissolve 0.7022 g 
Fe(NH 4 ) 2 (S0 4 ) 2 .6H 2 in H 2 0, add 1 ml_ H 2 S0 4 , and dil. to 1 L 

(d) Molybdenum std soins.— (7) Stock soln.— 100 i^g/mL. Dis- 
solve 0.0920 g (NH 4 ) 6 Mo 7 24 .4H 2 in H 2 and dil. to 500 mi_. (2) 
Working soln. — 5 /xg/mL. Dil. 25mL stock soln to 500 mL 

(e) Potassium thiocyanate soln. — 20%. Dissolve 50 g KSCN 
in H 2 and dil. to 250 mL 

(f) Sodium fluoride saturated soln.— Add 200 mL H 2 to ca 10 
g NaF. Stir until satd and filter. 

(g) Stannous chloride solns. — {/) 20% soln.— Weigh 10 g 
SnCI 2 .2H 2 into beaker, add 10 mL 20% HCI, (b)(7), and heat 
until completely dissolved. Cool, add granule of metallic Sn, dil. 
to 50 mL with H 2 0, and store in g-s bottle. {2) 0.8% wash soln.— 
Dil. 4 mL 20% soln to 100 mL with H 2 0. 



3058 



Determination 



[Caution: See 51.019, 51,026, and 51.028.) 

Weigh 1-5 g finely ground sample, contg ^35 (xg Mo, into 
200 mL tall-form Pyrex beaker. To 1, 2, or 5 g samples add 10, 
15, or 35 mL HN0 3 , resp. Include 2 beakers for blanks. Cover 
beaker with cover glass, and let stand ca 15 min; then heat 
cautiously on steam bath or hot plate at ca 100°, avoiding 
frothing overtop. If froth approaches cover glass, remove beaker 
from heat until frothing subsides; then continue heating. Digest, 
usually ca 2 hr, until most of solids disappear. 

Cool to room temp. If contents should go to dryness, add few 
mL HN0 3 . Add 6 mL 70-72% HCI0 4 , cover beaker, place on hot 
plate, and gradually raise temp, so that contents boil vigorously 
but do not bump. Continue heating until digestion is complete 
as indicated by liq. becoming colorless or pale yellow. If nec- 
essary, make repeated addns of HN0 3 and HCI0 4 and continue 
to digest until C is completely oxidized. 

After digestion is complete, place cover glass slightly to one 
side of top of beaker, or replace it with elevated watch glass, 
and evap. just to dryness or until residue appears only slightly 
moist. Remove beaker from hot plate, and cool. Wash down 
sides of beaker and underside of cover glass with few mL H 2 0, 
return to hot plate, and boil few min. Remove from hot plate, 
cool, and again rinse sides of beaker and cover glass with small 
amt H 2 0. 

Add 2 drops Me orange and neutze with NH 4 OH. Add 6/V HCI, 
dropwise with stirring, until soln is just acid; then add 8.2 mL 
excess to give final concn of ca 3% HCI. Add 2 mL satd NaF 
soln, and 1 mL Fe soln, if sample contains <100 /xg Fe. 

Transfer soln to 1 25 mL separator and dil. to 50 mL with H 2 0. 
Add 4 mL 20% KSCN soln, mix thoroly, and add 1.5 mL 20% 
SnCl 2 soln. Mix again, and from buret or pipet, add exactly 15 
mL isoamyi alcohol. Stopper separator and shake vigorously 1 
min, let phases sep., and drain and discard aq. layer. Ext into 
alcohol without delay, since colored complex is somewhat 
unstable in aq. soln. 

Add 25 mL freshly prepd 0.8% SnCI 2 wash soln, and shake 
gently 15 sec. Let phases sep., and drain and discard aq. layer. 
Transfer isoamyi alcohol soln to centrf. tube, and centrf. 5 min 
at ca 2000 rpm to remove H 2 droplets. If alcohol layer is not 
clear, recentrf. Stopper tubes to prevent evapn, if A readings 
cannot be made immediately. 

Compare unknown solns with isoamyi alcohol at ca 465 nm 



in spectrophtr, and make appropriate corrections in A readings 
for Mo in blanks. Obtain Mo concn from calibration curve 
relating A readings to conens of series of solns of known Mo 
content. 

Prep, calibration curve for instrument used, as follows: Place 
aliquots of working std soln contg 0, 5, 10, 15, 20, 25, 30, and 
35 /xg Mo, resp., into 200 mL tall-form beakers and carry them 
thru entire detn, beginning with digestion with HN0 3 and HCI0 4 . 
Plot A against corresponding Mo conens. 



NONMETALS 
Arsenic — Official Final Action 
3.059 Preparation of Solution 

See 25.008. 



3.060 



Determination 



Proceed as in 25.009, or take aliquot and det. as in 6.013, 
beginning ". . . add 3 mL H 2 S0 4 , . . ." 

Sulfur — Official Final Action 

Sodium Peroxide Method [18) 

{Caution: See 51.035.) 

3.061 Preparation of Solution 

Place 1.5-2.5 g sample in ca 100 mL Ni crucible and add 5 g 
anhyd. Na 2 C0 3 . Mix thoroly, using Ni or Pt rod, and moisten 
with ca 2 mL H 2 0. Add Na 2 2 , ca 0.5 g at time, mixing thoroly 
after each addn, and continue until mixt. becomes nearly dry 
and quite granular (ca 5 g Na 2 2 ). Place crucible over S-free flame 
or elec. hot plate and heat carefully, stirring occasionally, until 
contents are fused. (If material ignites, detn is worthless.) 

After fusion, remove crucible, let cool somewhat, and cover 
hardened mass with more Na 2 2 to depth of ca 5 mm. Heat 
gradually and finally with full flame until fusion again takes 
place, rotating crucible occasionally to bring any particles ad- 
hering to sides into contact with oxidizing material. Continue 
heating 10 min after fusion is complete. Cool somewhat, place 
warm crucible and contents in 600 mL beaker, and carefully add 
ca 100 mL H 2 0. After initial violent action ceases, wash material 
out of crucible, make slightly acid with HCI (adding small 
portions at time), transfer to 500 mL vol. flask, cool, dil. to vol., 
and filter. 



3.062 



Determination 



Dil. aliquot of prepd soln to ca 200 mL with H 2 and add HCI 
until ca 0.5 mL free acid is present. Heat to bp and add 10 mL 
1 0% BaCI 2 soln dropwise with constant stirring. Continue boiling 
ca 5 min, and let stand ^5 hr in warm place. Decant thru ashless 
paper or ignited and weighed gooch. Add 15-20 mL boiling H 2 
to ppt, transfer to filter, and wash with boiling H 2 until filtrate 
is Cl-free. Dry ppt and filter, ignite, and weigh as BaS0 4 . Wt ppt 
x 0.1374 = S. 

Magnesium Nitrate Method {19) 

3.063 Preparation of Solution 

Weigh 1 g sample into large porcelain crucible. Add 7.5 mL 
Mg(N0 3 ) 2 soln, 2.019, so that all material comes in contact with 
soln. (It is important that enough Mg(N0 3 ) 2 soln be added to 
ensure complete oxidn and fixation of the S present. For larger 
samples and for samples with high S content, proportionally 
larger vol. of this soln must be used.) Heat on elec. hot plate 



AOAC Methods (1980) 



Chlorine 



39 



(180°) until no further action occurs. Transfer crucible while hot 
to furnace (=^500°) and let it remain until sample is thoroly 
oxidized. (No black particles should remain. If necessary, break 
up sample and return to furnace.) Remove crucible and let cool. 
Add H 2 0; then HCI in excess. Bring soln to boil, filter, and wash 
thoroly. If preferred, transfer soln to 250 mL vol. flask before 
filtering and dil. to vol. with H 2 0. 



3.064 



Determination 



Dil. entire filtered soln, 3.063, to 200 mL, or take 100 mL 
aliquot of the measured vol., dil. to 200 mL, and proceed as in 
3.062. 



Gravimetric Quinolinium Molybdophosphate 
Method (22) — Official Final Action 

3.069 Preparation of Solution 

Accurately weigh ca 2 g plant sample in porcelain dish, and 
add 7.5 mL Mg(N0 3 ) 2 soln, 2.019. Dry in oven 2 hr at 110-115° 
(or until dry). Ignite carefully over Fisher burner, or equiv., until 
bubbling and smoking cease. Complete ashing in furnace 4 hr 
at 550-600°. Dissolve ash in few mL HCI (2+1) and evap. to 
dryness on steam bath. Take up residue in 10-15 mL HCI (1+9) 
and filter thru coarse paper into 200 mL vol. flask. Wash paper 
thoroly with H 2 and let filtrate cool to room temp. Dil. to vol. 
with H 2 0. 



Phosphorus (20) — Official Final Action 
3.065 * Macro Method * 

(a) For samples exceedingly high in P and low in Ca and Mg 
{certain seeds, grains, etc.) — Prep, soln as in 3.063, or evap. 
filtrate and washings from S detn, 3.062, to 50 mL, and proceed 
as in 8.033. 

(b) For other samples. — Take 50 mL aliquot of Soln /, 3.005, 
and proceed as in 8.033. 



3.070 



Determination 



Micro Method (27) 



3.066 



Reagents 



(a) Phosphorus std soln.— 0.025 mg P/mL. Dissolve 0.4394 g 
pure dry KH 2 P0 4 in H 2 and dil. to 1 L. Dil. 50 mL of this soln to 
200 mL. 

(b) Ammonium molybdate soln. — Dissolve 25 g NH 4 moly fa- 
date in 300 mL H 2 0. Dil. 75 mL H 2 S0 4 to 200 mL and add to NH 4 
molybdate soln. 

(c) Hydroquinone soln. — Dissolve 0.5 g hydroquinone in 100 
mL H 2 0, and add 1 drop H 2 S0 4 to retard oxidn. 

(d) Sodium sulfite soln.— Dissolve 200 g Na 2 S0 3 in H 2 0, dil. to 
1 L, and filter. Either keep this soln well stoppered or prep, fresh 
each time. 



3.067 



Preparation of Solution 



To 1 or 2 g sample in small porcelain crucible add 1 mL 
Mg(N0 3 ) 2 soln, 2.019, and place on steam bath. After few min, 
cautiously add few drops HCI, taking care that gas evolution 
does not push portions of sample over edge of crucible. Make 
2 or 3 further addns of few drops HCI while sample is on bath 
so that as it approaches dryness it tends to char. If contents 
become too viscous for further drying on bath, complete drying 
on hot plate. Cover crucible, transfer to cold furnace, and ignite 
6 hr at 500°, or until even gray ash is obtained. (If necessary, 
cool crucible, dissolve ash in little H 2 or alc.-glycerol, evap. to 
dryness, and return uncovered to furnace 4-5 hr longer.) Cool, 
take up with HCI (1+4), and transfer to 100 mL beaker. Add 5 
mL HCI and evap. to dryness on steam bath to dehydrate Si0 2 . 
Moisten residue with 2 mL HCI, add ca 50 mL H 2 0, and heat few 
min on bath. Transfer to 100 mL vol. flask, cool immediately, 
dil. to vol., mix, and filter, discarding first portion of filtrate. 



3.068 



Determination 



To 5 mL aliquot filtrate in 10 mL vol. flask add 1 mL NH 4 
molybdate soln, rotate flask to mix, and let stand few sec. Add 
1 mL hydroquinone soln, again rotate flask, and add 1 mL Na 2 S0 3 
soln. (Last 3 addns may be made with Mohr pipet.) Dil. to vol. 
with H 2 0, stopper flask with thumb or forefinger, and shake to 
mix thoroly. Let stand 30 min, and measured with spectrophtr 
set at 650 nm. Report as % P. 



Pipet 40 mL aliquot into 300 or 500 mL erlenmeyer and 
proceed as in 2.028. 

Chlorine (23) — Official Final Action 

(If bromides or iodides are present in significant 
amts, correct results accordingly.) 

3.071 Preparation of Solution 

Verify complete retention of CI in each kind of material by 
trial, since losses can occur, especially with samples high in 
carbohydrates, if insufficient Na 2 C0 3 is present during ignition, 
or in any case if excessive temp, is used. 

Moisten 5 g sample in Pt dish with 20 mL 5% Na 2 C0 3 soln, 
evap. to dryness, and ignite as thoroly as possible at ^500°. Ext 
with hot H 2 0, filter, and wash. Return residue to Pt dish and 
ignite to ash; dissolve in HN0 3 (1+4), filter, wash thoroly, and 
add this soln to H 2 ext. 



3,072 



Gravimetric Method 



To prepd soln, add 10% AgN0 3 , avoiding more than slight 
excess. Heat to bp, protect from light, and let stand until ppt 
coagulates. Filter on weighed gooch, previously heated to 
140-150°, and wash with hot H 2 0, testing filtrate to prove excess 
of AgN0 3 . Dry AgCI at 140-150°, cool, and weigh. Report as % 
CI. 

Volumetric Method I (24) 

(Since precision of this titrn is considered to be ±0.2 mg CI, 
accuracy of 1.0% requires samples contg 5*20 mg.) 



3.073 



Reagents 



(a) Silver nitrate std soln.—l mL - 0.00355 g CI. Prep, soln 
slightly stronger than 0.1/V, stdze as in 50.031, and adjust to 
exactly 0.1/V. 

(b) Ammonium or potassium thiocyanate std soln. — 0.1/V. 
Prep, soln slightly stronger than 0.1/V, stdze as in 50.030(b), and 
adjust to exactly 0.1/V. 

(c) Ferric indicator.— Satd soln of FeNH 4 (S0 4 ) 2 .12H 2 0. 

(d) Nitric acid. — Free from lower oxides of N by dilg HN0 3 
with ca Va vol. H 2 0, and boiling until perfectly colorless. 



3.074 



Determination 



To prepd soln, 3.071, add known vol. std AgN0 3 soln in slight 
excess. Stir well, filter, and wash AgCI ppt thoroly. To combined 
filtrate and washings add 5 mL ferric indicator and few mL 
HN0 3 , and tttr. excess Ag with thiocyanate std soln to permanent 
light brown. From mL AgN0 3 used, calc. % CI. 

ir Surplus method— see inside front cover. 



40 



3. Plants 



AOAC Methods (1980) 



Volumetric Method II &5) 
3.075 Reagents 

(a) Potassium iodide std so In. — 1 mL=1 mg CI. Weigh 4.6824 
g pure (ACS) Kl, dried to const wt at 105-150°, dissolve in H 2 0, 
and dil. to 1 L. 

(b) Silver nitrate stock so In. — Approx. 0.3/V. 1 mL = ca 10 mg 
CI. Dissolve 48 g AgN0 3 in H 2 0, filter, and dil. to 1 L. 

(c) Silver nitrate std soln. — Dil. 100 mL reagent (b) to ca 900 
mL and adjust by stdzg against reagent (a) so that 1 mL = 1 mg 
CI. 

(d) Chloride-free starch indicator. — For each 100 mL final soln 
take 2.5 g sol. starch and make to paste with cold H 2 0. Stir out 
lumps, add 25-50 mL more cold H 2 0, and stir or shake 5 min. 
Centrf., decant, and discard liq. Repeat extn 3 times and finally 
transfer residue to flask contg proper amt of boiling H 2 0. Stir 
again, heat to bp, cover with small beaker, and cool under tap, 
shaking occasionally. 

(e) Dilute sulfuric acid.— Add 35 mL H 2 S0 4 to each 1 L H 2 0, 
boil 5-10 min, and cool to room temp. 

(ff) Iodine indicator. — To ca 20 g I in 500 mL g-s bottle add 400 
mL dil. H 2 S0 4 , (e), and shake 10 min. Decant and discard first 
soln, since it may contain iodides. Repeat process and store 
soln in small g-s bottles. 

(g) Potassium permanganate soln. — Dissolve 60 g KMn0 4 in 
400 mL warm H 2 (ca 50°) and dil. to 1 L. 

(h) Potassium sulfate-copper sulfate mixture. — Thoroly mix 
16 parts K 2 S0 4 and 1 part CuS0 4 .5H 2 0. 

(i) Wash soln.— Mix 980 mL H 2 and 20 mL HN0 3 . 



3.076 



Determination 



{Caution: See 51.019, 51.026, and 51.080.) 



Weigh sample contg 10—40 mg CI into beaker. (If >4 g is taken, 
use proportionately more HN0 3 and KMn0 4 soln.) Add 10 mL 
0.3/V AgN0 3 and stir until sample is thoroly soaked, adding little 
H 2 or warming if necessary. Add 25 mL HN0 3 , stir, add 5 mL 
KMn0 4 soln, and stir until frothing stops. Place mixt. in H 2 
bath or on hot plate and keep just below bp. Stir, and wash 
down sides of beaker at intervals with min. H 2 0. After 20 min, 
or when reaction stops, add addnl KMn0 4 soln, little at time, 
until color begins to fade slowly. Dil. to ca 125 mL with boiling 
H 2 and heat 10 min longer. (Beaker may stand in bath or on 
hot plate until ready to filter.) 

Filter while hot thru Whatman No. 5, or equiv. paper, with 
suction as follows: Place disk of 30-mesh stainless steel wire 
gauze or No. 40 filter cloth in bottom of 3" (7.6 cm) Hirsch funnel. 
Fold 9 cm paper over bottom of No. 11 rubber stopper, shaping 
it to funnel by making 9-10 folds up side of stopper. Place paper 
in funnel and apply strong suction. Wet paper and keep wet 
while fitting into funnel so as to avoid double thicknesses of 
paper. Wash paper thoroly, first with H 2 and then with wash 
soln. Discard washings and rinse out flask. Decant thru filter 
and transfer ppt and sample residue to filter. If filtrate is not 
turbid, or if it is only slightly opalescent, wash ppt thoroly, 
applying wash soln very gently, but keeping strong suction on 
filter. If combined filtrate and washings are clear, test for Ag. If 
turbid, reheat and pass thru filter, repeating until clear, and 
finally wash as above. If filtrate does not give definite test for 
Ag, repeat detn on smaller sample. 

Place paper and contents in Kjeldahl flask and add such amts 
of K 2 S0 4 -CuS0 4 mixt. and H 2 S0 4 as would be appropriate for 
protein detn on same kind and amt of sample, and digest 
similarly. (For 2 g grass, 8 g sulfate mixt. and 20 mL acid are 
enough.) When digest is cool, add 175 mL H 2 0, boil 5-10 min, 
and cool to room temp. Titr. the Ag 2 S0 4 in Kjeldahl flask with Kl 



std soln, using 5 mL starch indicator and 30 mL I indicator. (Add 
latter just before titrn.) Rinse neck of flask after each addn of Kl 
when near end point and titr. until soln stays blue after shaking. 
If <30 mg CI is present, add starch and I solns at beginning. If 
larger but unknown amt is present, add 2 mL starch and 10 mL 
I indicator at beginning and titr. until end point approaches. 
Shake vigorously to coagulate ppt, add rest of starch and I solns, 
and proceed to end point. If known large amt is present, titr. to 
within 2 mL of end point, shake as above, add indicator reagents, 
and continue titrn. If end point is overrun, add 5 mL std AgN0 3 
soln and titr. again. 

Blank detns are not necessary after testing reagents. If blank 
made by using pure sugar as sample is >0.05 mg, examine 
filter paper, distd H 2 0, and various reagents for CI. 

Fluoride 
Potentiometric Method i26)—Official First Action 

(Rinse all plastic and glass containers with HCI (1+3) and H 2 

before use. Perform analyses in laboratory free from F; prep. 

samples in another laboratory.) 

3.077 Principle 

F is extd from dry, pulverized foliage with HN0 3 followed by 
aq. KOH, Slurry is adjusted to pH 5.5, and complexing agent and 
background F are added. Potential is measured with ion selective 
electrode and compared against calibration curve. Method is 
applicable to 10-2000 (xg F/g dry wt leaf tissue not exposed to 
unusual amts of Al or other F-binding agents; it is not applicable 
to insol. inorg. F or F in org. combinations. Between-laboratory 
precision of individual analyses is ±20% at 30 ppm F; ±10%, 
5*100 ppm F. Accuracy is 90-100%. 

3.078 Apparatus 

Electrometer. — Range ±200 mv with readability of 0.1 mv 
{Model 701 or 701 A digital pH/mv meter, Orion Research Inc., 
or equiv.) or expanded scale pH meter with mv mode of 
operation, with F ion selective electrode (No. 94-09 single 
electrode, Orion Research Inc., or equiv.) and reference electrode 
(No. 90-01 single junction, Orion Research Inc., or equiv.). Check 
system at intervals to assure adherence to following perform- 
ance criteria: Using technic of 3.080, system should reach 
equilibrium (AE <0.2 mv/min) within 5 min with each F working 
std soln, checked in following sequence: 0.1, 0.2, 0.5, 2.0, and 
10.0 ppm F. Replicate std solns should differ by =£1 mv. Cali- 
bration curve should be linear between 0.2 and 10.0 ppm and 
slope should be 57±2 mv per 10-fold change in F concn. If any 
parameter is not obtained, check electrodes, reagents, and 
electrometer. Maintain temp, control to ±1°. 

3.079 Reagents 

(Store all solns in tightly closed, plastic bottles.) 

(a) Nitric acid.— {7) /0/V.™ Add 63 mL HN0 3 to H 2 0, cool, and 
dil. to 100 mL. (2) 0.2N.—DW. 5.0 mL 10/V to 250 mL. (3) 0.05N.— 
Dil. 5.0 mL 10/V to 1 L 

(b) Potassium nitrate soln. — 0.4M. Dissolve 4.0 g KN0 3 in H 2 
and dil. to 100 mL. 

(c) Sodium citrate soln.— 0.8/W. Dissolve 58.8 g Na citrate. 2H 2 
in 200 mL H 2 0, adjust to pH 5.5 by dropwise addn of 10/V HN0 3 , 
using pH meter, and dil. to 250 mL with H 2 0. 

(d) Sodium citrate with fluoride soln. — 0.4M citrate with 1 ppm 
F. Dil. 125 mL 0.8/W Na citrate soln and 25.0 mL 10 ppm F std 
soln to 250 mL with H 2 0. 

(e) Fluoride std solns. — (7) Stock soln. — 100 ppm F, Dry ca 1 
g NaF 2 hr at 110°. Accurately weigh 0.221 g NaF, dissolve in 



AOAC Methods (1980) 



Fluoride 



41 



Table 3:01 Preparation of Working Standard Solutions 







mL soln to be dild to 100 nr 


L 




Concn, 


0.4/W 


0.8M Na 


100 ppm 




10 ppm 


ppm 


KN0 3 


citrate 


F soln 




F soln 


10 


10.0 


5.0 


10.0 




0.0 


2 


10.0 


5.0 


2.0 




0.0 


0.5 


10.0 


5.0 


0.0 




5.0 


0.2 


10.0 


5.0 


0.0 




2.0 


0.1 


10.0 


10.0 mL Na citrate soln 


contg 1 


ppm F 



H 2 0, and dil. to 1 L. (2) Intermediate soln.— 10 ppm F. Dil. 10.0 
mL stock soln to 100 mL with H 2 0. {3) Working solns. — Prep, as 
in Tabfe 3:01 in 100 mL vol. flasks. Prep. 0.2 and 0.1 ppm solns 
fresh as needed. 



3.080 



Preparation of Calibration Curve 



Place 25.0 mL 0.1 ppm F working std soln into plastic container 
contg stirring bar. Insert electrodes ca 12 mm into soln and stir 
mag. Record mv readings at 1 min intervals until change is <0.2 
mv/min. Remove electrodes, blot lightly with absorbent paper, 
and repeat reading with 0.2, 0.5, 2.0, and 10.0 ppm std solns. 
Place electrodes in 0.2 ppm std soln until samples are analyzed. 
(10 ppm std soln may be omitted if samples are known to 
contain <400 ppm F.) 

Plot potential (mv) on vertical arithmetic axis and F concn 
{fig/ mL; ppm) on horizontal (logarithmic) axis of 2-cycle semilog 
graph paper. 

3.081 Preparation of Sample 

Dry foliage 48 hr at 80°. Grind to pass No. 40 sieve and store 
in clean, dry, tightly closed plastic bottle. Rotate bottle to mix 
sample thoroly before removing aliquots. 



3.082 



Determination 



Accurately weigh ca 0.25 g powd sample, and place in 75-100 
mL wide-mouth plastic container. Add 20 mL 0.05A/ HN0 3 and 
place on rotating shaker or stir mag. 20 min. Add 20 mL 0.1 N 
KOH (5.6 g/L) and agitate addn) 20 min. Add 5.0 mL Na citrate 
soln contg 1 ppm F, adjusted to pH 5.5, and 5.0 mL 0.2/V HN0 3 . 



(Samples may be stored covered *s4 hr at this point.) Det. mv 
readings as in 3.080 and prep, calibration curve before and after 
each series of samples. 

If sample series contains mixt. of high and low samples, make 
preliminary estn of F content after 2 min. Then det. F concn in 
samples contg <40 ppm first and in higher ones last. 

ppm F (^g/g) = (C - 0.10) x 50/iv, 
where C = ppm F from curve; 0.1 = ppm background F in final 
soln; 50 = mL final soln; and w = g sample. 

ASTM-lntersociety Committee-AOAC 
Semiautomated Method {27) 

Official First Action 



3.083 



Principle 



Dried and ground plant material is ashed, fused with alkali, 
and dild to vol. In case of leaf samples, F on external surfaces 
may be washed off sep. Digest and H 2 S0 4 are pumped into 
microdistn app. maintained at 170°. Stream of air carries acidified 
sample to fractionation column where F and H 2 are distd into 
condenser, and condensate passes into small collector. Distillate 
is mixed continuously with alizarin F blue-lanthanum reagent, 
colored stream passes thru tubular flowcell of colorimeter, and 
A is measured at 624 nm. 

Interfering metal cations and inorg. phosphate are not distd, 
and org. substances are destroyed by ashing. Interference from 
remaining volatile inorg. anions is reduced with high concn 
acetate buffer with some reduction in sensitivity. Very large 
amts solid matter, particularly silicates, retard distn. Therefore, 
smallest sample consistent with obtaining suitable amt F should 
be used. Conditions must be carefully controlled, since accurate 
results depend upon obtaining same degree of efficiency of 
distn from samples as from std F solns used for calibration. 

Acid concn during distn is maintained at const value by 
using specific amts CaO and NaOH for ashing and fusion and 
HCI0 4 for transfer of fused samples. Any marked change in vac. 
(>0.2" Hg or 5 torr) over short time indicates either leak or block 
in system. Distil at same vac. each day and maintain proper 
ratio between air flow on line drawing liq. and solid wastes from 
distn coil and on line drawing HF and H 2 vapor from distn unit 




FIG. 3:02 — Schematic drawing of air flow system used in semiautomated analysis for fluoride 



42 



3. Plants 



AOAC Methods (1980) 



(Fig. 3:02). Adjust flowmeters to keep this ratio const and to 
maintain higher vac. on HF line, C n so that min. is diverted to 
waste line. 

Method can detect 0.1 jag F/mL. Normal range is 0.1-4.0 fxg 
F/mL Dil. higher concns with NaOH-HCI0 4 soln, (k). If digested 
samples routinely exceed 4.0 /xg/mL, modify anal, portion of 
pump manifold to reduce sensitivity, or use smaller sample 
aliquot (preferred). Most accurate results are obtained in middle 
or upper part of calibration curve. For example, to decrease 
sensitivity, pump sample thru 0.081" tube (2.5 mL/min) and dil. 
with H 2 pumped thru 0.065" tube (1.6 mL/min) before sample 
enters distn app. Total vol. sample and diluent should approx. 
original vol. used (4.1 mL/min). 

If air-borne contaminants are present in laboratory, attach 
small drying bulb contg CaC0 3 granules to air inlet tube of 
microdistn unit. Teflon distn coil of microdistn unit must be 
cleaned periodically to avoid accumulation of solids which 
reduce sensitivity. 

Coefficient of variation of 20-100 ppm F is generally ^10%. 
Samples with large amt of Si (orchard grass) or Al may present 
special difficulties. There should be no significant deviation from 
linearity with different amts sample and with different amts 
added F. 



3.084 



Apparatus 



(Cat. Nos refer to current Technicon equipment, except where 

indicated. Corresponding equipment under previous Cat. Nos 

is satisfactory.) 

(a) Automatic analyzer. — (Fig. 3:03) AutoAnalyzer, Technicon 
Instruments Corp., or equiv. (1) Sampler. — Sampler IV with 
rotary stirrer and 8.5 mL plastic sample cups. Use 10 or 20/hr 
cam with 1:3 sample-to-wash ratio (No. 171-A015-07). (2) Col- 
orimeter. — With 15 mm tubular flowcell and 624 nm interference 
filter (199-A001-05). (3) Recorder. —Ratio type with 2-100 mv 
full scale range (01 1-A1 15-01). [4] Multichannel proportioning 
pump and manifold cartridge. — With assorted pump tubes, 



nipple connectors, and glass connectors (pump III 113-A014-08; 
cartridge 116-8340-01). 

(b) Pulse suppressors. — For sample and color reagent 
streams. Coil 10' length of 0.035" id Teflon std tubing around 
2.5" diam. tube. Force outlet end into short length of 0.045" id 
Tygon tubing which is then sleeved with piece of 0.081" Tygon 
tubing. Slip sleeved end over "h" fitting which joins sample and 
reagent streams. (Pulse suppressor included with manifold 
cartridge.) 

(c) Voltage ste/?/7/zer.— 161-A007-01 (also part of 199-A001- 
05). 

(d) Rotary vacuum and pressure pump. — With continuous 
oiler (Gast No. 0211-V45F-G8CX pump, available from SGA 
Scientific, Inc.). 

(e) Microdistillation apparatus. — (Fig. 3:04) Major compo- 
nents are (Cat. Nos. are those of SGA Scientific, Inc., except as 
noted): (7) Bottom only of jacketed 1 L resin reaction flask with 
conical flange (JR-5130), modified by evacuating space between 
inner and outer walls and sealing off port (a); (2) resin reaction 
flask top with conical flange (JR-7935) modified to have one f 
29/42 center joint and four? 24/40 side joints; (3) resin reaction 
flask clamp (JR-9210); (4) variable high-speed stirrer (S-6362) {d); 
(5) stainless steel, heavy duty stirrer stuffing box with ¥ 29/42 
and shredded Teflon packing (JS-1160and JS-3050); (6) 10 mm 
diam. stainless steel stirrer rod with propeller to fit stuffing box; 
(7) thermometer-thermoregulator, range 0-200° (T-5715) (c); (8) 
electronic relay control box (T-5905); {9) low drift immersion 
heater, 750 watts (H-1265) (b); {10) 30' length coil of flexible 
Teflon TFE tubing, 1 / 8 " id, 3/16" od, 0.030" wall, on rigid support 
of such diam. that completed coil will fit into resin reaction flask 
(avoid kinking of tubing) (e); {11) 2 flowmeters with ranges 0-1 
and 0-5 L/min, both with needle valve controls (Dwyer Instru- 
ment, Inc., PO Box 373, Michigan City, IN 46360); {12) vac. gage 
with range 0-10" Hg or 0-254 torr (mm Hg); {13) fractionation 
column of borosilicate glass {g; see also Fig. 3:05; 116-0635); 
{14) distillate collector (B2 fitting; 116-011-01); (75) H 2 0-jacketed 
condenser (116-0156-01) (/?); {16) Dow-Corning 200 fluid (100 
centistokes at 25°) (f); and (17) condenser (116-0181-01) (/). 



Iuppc, 
L o~ ef|Level 

Level | 



Tube Size 
(inches) Sampler 11 



DO 






(7) 0.090 Water (2.90 ml/min) 



®0. 



056 Water (1.20 ml/min) 



-®= 



@ 0O90Sample (2.90 ml/min) 




-O 



0.035 Air (0.42ml/min) 



Microdistillation 
device 



® 




© 0.081 Acid t 250 ml/min) 



(J) 0.081 Impinger (2.50 ml/min) 



@ 0.073(5) A-C 
H ) Reagent (1.69 ml/ min) 

0.045 (S) (0.70 ml/min) 



Vacuum 
pump 



COLORIMETER RECORDER 

'5 mm Tubular f c 
6 24 m ,, Filters 



RG. 3:03 — Flow diagram for semiautomated analysis for fluoride 



AOAC Methods (1980) 



Fluoride 



43 




FIG. 3:04 — Schematic drawing of microdistillation apparatus 

(f) Crucibles. — Inconel, Ni, or Pt, 40-50 mL. 

(g) Air flow system. — (Fig. 3:02) Draw air thru air inlet tube, 
(a), before Teflon microdistn coil, (/?). Air sweeps thru {b) to 
fractionation column, and is diverted into 2 channels. In 
channel c v air passes thru H 2 0-jacketed condenser, (d), sample 
trap, (e), to waste bottle, [f). Air then passes thru %" id glass 
tube directed against surface of H 2 S0 4 in waste bottle, (g). 
Partially dehydrated air passes thru gas drying tower, {h) t contg 
450 g indicating silica gel. Emerging air passes thru T-tube, (/), 
connected to vac. gage, (/) (0-10" Hg or 0-254 torr), thru 
flowmeter, {k) (0-5 L/min), thru T-tube, {/), and then to vac. 
pump, (m). 

In channel c 2 , air passes thru H 2 0-jacketed waste trap, (/?), to 
waste bottle, (o). Air leaving waste bottle flows thru drying bulb, 
{p), filled with indicating silica gel, and the dry air then passes 
thru flowmeter, (g) (0-1 L/min). Air stream then connects thru 
T-tube, (/), with air from first channel. 



3.085 



Reagents 



{Caution: See 51.028 and 51.030.) 

(Deionized H 2 may be used. CaO for ashing and NaOH for 
fusion must be low in F.) 

(a) Sulfuric acid.— (1 +1). Mix 500 mL H 2 S0 4 with 500 mL H 2 
and cool before use. 

(b) Acetate buffer.— 2. 14/W(pH 4.0). Dissolve 60 gNaOAc.3H 2 
in 500 mL H 2 0, add 100 mL HOAc, and dil. to 1 L with H 2 0. Stable 
at 25°. 

(c) Alizarin fluorine blue color reagent stock soln. — 0.01/W. 
Suspend 0.9634 g reagent (alizarin complexone, alizarin com- 
plexan; 3-amino-ethy(alizarin-/V,/V-diacetic acid; Burdtck & Jack- 
son Laboratories, Inc.) in ca 100 mL H 2 in 250 mL vol. flask. 
Add 2 mL NH 4 OH and shake until completely dissolved. Add 2 
mL HOAc and dil. to vol. with H 2 0. Stable indefinitely at 4°. 

(d) Lanthanum nitrate stock soln. — 0.02M. Dissolve 8.6608 g 
La(N0 3 ) 3 .6H 2 in H 2 and dil. to 1 L in vol. flask. 

(e) Wetting soln. — 30% soln (w/v) polyoxy ethylene lauryl 
ether in H 2 (Brij-35, Technicon No. T21-0110). Soln is stable at 
25°. 

(f) Working reagent. — Mix, in order listed, 300 mL acetate 




16. 5 cm 



shoulder should be 
rounded, not abrupt 



mm D 



FIG. 3:05 — Microdistillation column 



buffer, 244 mL H 2 0, 300 mL acetone, 100 mL tert-butanol, 36 mL 
alizarin fluorine blue stock soln, 20 mL La (N0 3 ) 3 stock soln, and 
2 mL wetting soln. Unused reagent is stable 7 days at 4°. Before 
using reagent, place under vac. 10 min to remove air bubbles 
from soln. 

(g) Fluoride std solns. — (7) Stock soln. — 100 /jlq F/mL. Dis- 
solve 0.2207 g NaF in H 2 and dil. to 1 L. {2) Working solns.— 
Prep. 7 solns contg 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, and 4.0 fxg F/mL. 
Before dilg to vol., add 6 g NaOH and 20 mL 70% HCI0 4 for each 
100 mL final working soln so that stds have same composition 
as sample solns. Dil. with H 2 only for analysis of H 2 samples 
or air samples absorbed in H 2 0. Store working solns at 4° in 
polyethylene bottles; stable in presence of NaOH. 

(h) EDTA solns.— 1%. Dissolve 1 g Na 4 EDTA in 99 mL H 2 0. 
Prep. 0.05% and 0.01% solns by mixing 5 mL 1% soln with 95 
mL H 2 and 1 mL 1% soln with 99 mL H 2 0, resp. 

(i) Phenolphthalein soln. — Dissolve 1 g phthln in 50 mL ab- 
solute alcohol or isopropanol and add 50 mL H 2 0. 

(j) Detergent. — Alconox (A Icon ox, Inc., 215 Park Ave S, New 
York, NY 10003); available from laboratory supply firms. 

(k) Sodium hydroxide-perchloric acid soln. — Dissolve 6 g 
NaOH in H 2 0, add 40 mL 70% HCI0 4 (1+1), and dil. to 100 mL 
with H 2 0. Use to dil. samples when F in unknown sample 
exceeds std curve. 



3.086 



Preparation of Sampfe 



(a) Leaves. — If it is necessary to remove surface F, wash 
sample with aq. soln contg 0.05% detergent and 0.05% Na 4 EDTA 
in polyethylene container 30 sec with gentle agitation. Remove, 
drain 3-4 sec, and rinse 10 sec in each of 3 beakers of H 2 0. 
Discard solns after use. 

(b) Fresh plant tissues. — Dry 24-48 hr in 80° forced-draft oven, 
and grind as in (c). 

(c) Dry plant tissues. — Grind in semimicro Wiley mill to pass 
No. 40 sieve, and store in plastic container. 

3.087 Ashing and Fusion 

Accurately weigh 0.1-2.0 g well mixed dried plant tissue into 
crucible. Add 100±10 mg low-F CaO, enough H 2 to make loose 
slurry, and 4 drops phthln soln. Mix thoroly with polyethylene 
policeman. Final mixt. should be purple and remain purple 
during evapn to dryness. 



44 



3. Plants 



AOAC Methods (1980) 



Place crucible on cold hot plate and under IR lamp. Evap. 
under lamp to dryness, turn on hot plate, and char 1 hr. Transfer 
crucible to furnace at 600° and ash 2 hr. {Caution: To avoid 
flaming, place crucibles at front of furnace with door open ca 
5 min to further char samples; then reposition in furnace.) 

Remove crucibles, add 3.0±0.1 g NaOH pellets, and replace 
in furnace with door closed to melt NaOH. {Caution: Avoid 
creeping of molten NaOH.) Remove crucibles individually and 
swirl to suspend particulate matter until melt is partially solid- 
ified. Let cool until addn of small amt H 2 does not cause 
spattering. Wash down inner walls with 10-15 mLH 2 0. Suspend 
melt with polyethylene policeman and transfer with H 2 to 
plastic tube graduated at 50 mL. Rinse crucible with 20.0 mL 
70% HCI0 4 (1 + 1), add rinse to tube, and dil. to 50 mL with H 2 0. 
Solns can be stored at this point if tightly capped. 

Analyze blank contg all reagents with each set of ca 10 
samples. 

Clean crucibles as soon as possible after each use. Boil Inconel 
crucibles 1 hr in 10% NaOH soln. Rinse with hot tap H 2 0, 
detergent, and then distd H 2 0. Immerse crucibles which held 
samples contg >100 ^9 F in 4A/ HCI 45 min before boiling in 
NaOH soln. Perform blank analyses on these crucibles before 
addnl use to check for contamination. Scrub Ni and Pt crucibles 
with detergent and hot H 2 and rinse thoroly with H 2 0. Briefly 
rinse crucibles which held samples contg >100 fig F in 4/V HCI 
before rinsing with H 2 0. 

3.088 Analytical System 

Place F std solns, ashed and fused samples, or impinged air 
samples in 8.5 mL plastic cups in sample module. Actuate 
sampler and pump from cup at net rate of 2.48 mL/min with air 
segmentation of 0.42 mL/min after sampler crook, and pump 
into microdistn device thru sample inlet (/, Fig. 3:04), using 
0.051" id Teflon tubing. Pump H 2 S0 4 at 2.5 mL/min thru acid 
inlet {m, Fig. 3:04). Cool and discard acid and solids. Pump 
distillate from sample trap at 2.0 mL/min thru 0.051" Teflon 
tubing, add color reagent at 1.69 mL/min, and mix in 4" length 
of Vs" id glass tubing packed with pieces of 20 mesh broken 
Pyrex glass. Pass colored stream thru time delay coil of 15' of 
0.035" Teflon spaghetti tubing, thru debubbler fitting where 
small portion of stream and bubbles are removed to waste 
bottle at rate of 0.70 mL/min, and thru 15 mm tubular flowcell 
of colorimeter. A is measured at 624 nm and plotted on recorder. 
Lag time from sampling to appearance of peak is ca 5 min. Time 
between samples is 6 min with sampling rate of 10/ hr and 3 
min at 20/ hr. 

3.089 Start-Up 

Turn on H 2 to condenser and cooling jacket. Turn on color- 
imeter. Engage manifold on proportioning pump and start 
pump. Turn on stirring motor of microdistn unit, vac. pump 
adjusted for full vac, and heater of microdistn unit. Connect 
lines to H 2 S0 4 , color reagent, and H 2 bottles. Sampling tube of 
sampler unit should be in H 2 reservoir. Equilibrate app. until 
silicone oil in microdistn unit reaches 170±2°. Check that all 
connections are secure. Adjust distn flowmeter {k, Fig. 3:02) to 
2.5-3 L/min; adjust waste flowmeter (q) to 0.3 L/min. Distillate 
should now fill sample trap. Readjust flowmeter {k) to give 
reading on vac. gage of 5-6" Hg (127-150 torr). (Satisfactory 
setting for app. must be detd by trial and error. Once detd, use 
each day.) No air bubbles should be in anal, system beyond 
point where color reagent and distillate streams are joined. Turn 
on recorder, adjust baseline to desired level, and run several 
min to assure that all components are operating properly. 
Baseline should be reasonably smooth and straight. 



Transfer F std solns to 8.5 mL plastic cups and place in 
sampler. Sep. last std soln from sample solns with cup of H 2 0. 
Program sampler for 10 samples/hr {90 sec sampling period, 
270 sec washout period) or 20 samples/hr (45 sec sampling 
period, 135 sec washout period). 

Prep, std curve, 3.092, before and after each day's set of 
samples. Net A of 0.7-0.9 should be obtained with std soln 
contg 4 fxg F/mL. A of each std soln should be reproducible 
within 10% from day to day and std curve should be linear from 
0.2 to =*3.2 /xg/mL 



3.090 



Shut-Down 



Turn off chart recorder. Disconnect H 2 S0 4 line and place in 
H 2 0. Disconnect color reagent line and place in 0.01% EDTA 
soln ca 1 min; then transfer line to H 2 and let H 2 pass thru 
system ca 5 min. Clean Teflon distn coil as in 3.091(a). Turn off 
heater and stirrer of microdistn unit. Turn off vac. pump. Release 
pump tube manifold. Turn off H 2 to condenser and cooling 
trap. 



3.091 



Maintenance 



(a) Cleaning of Teflon distillation coil. — (After use with sam- 
ples contg particulate matter.) Briefly insert Tygon tube con- 
nected to air inlet line of microdistn unit into 0.01% EDTA soln. 
After all deposited material has been removed, wash with 3-4 
five mL portions distd H 2 0. 

(b) Pump tubes.— Replace after 200 working hr or earlier if 
hard and inflexible or flattened. Always leave in relaxed position 
when not in use. Remove dirt and grease from pump plates and 
rollers after each day of use. 

(c) Indicating silica gel. — Regenerate when ca 2 A has lost 
normal blue color. 

(d) Cleaning tubing.— Clean tubing contg reagent after each 
daily run with 0.01% EDTA soln followed by distd H 2 0. 

(e) Monthly checks. — Oil proportioning pumps monthly. 
Check gain on recorder monthly and adjust. 



3.092 



Calibration and Standards 



Before and after each day's set of samples, prep, std curve by 
transferring aliquots of each working std soln to 8.5 mL sample 
cups and proceed with analysis. Draw straight line connecting 
baseline before and after analysis. Records of each peak and 
subtract A of baseline at peak. Plot net>4 against jug F/mL. 

3093 Calculations 

ppm F in sample = (F x V x D)/W, 
where F = ^g F/mL sample from std curve; V = mL sample, 
usually 50; D = diln factor used only when F of sample exceeds 
std curve = mL final vol. to which original aliquot was dild with 
NaOH-HCI0 4 soln, (k)/mL original aliquot taken; and W = g 
sample taken for analysis. 



3.094 



Check Procedure 



(a) Contamination. — Perform reagent and equipment blank 
with crucibles and reagents but without sample to detect con- 
tamination from previous samples, contaminated furnace, and 
reagents. Blank values >5 fxg F are evidence of contamination. 
Perform 2 blank detns with each set of 20-40 samples. Usual 
blanks are 1-3 /xg. 

(b) Recoveries. — Occasionally add known amts F std soln 
from microburet to aliquots of low F tissue. Recovery of added 
F should be 100±10%. Low values indicate loss of F, possibly 
during pretreatment; high values indicate contamination. 



AOAC Methods (1980) 



Selenium 



45 



(c) Linearity. — Occasionally analyze different amts (0.1-2.0 g) 
plant sample contg 50-65 ppm F. Linear relationship should 
exist between F found and amt tissue taken. Nonlinearity may 
indicate that some component of tissue is retarding distn or 
interfering with color development 

(d) Calibration curves. — Prep, at least twice daily. 

3.095 Trouble Shooting 

(a) Irregular baseline. — May result from: {1) excessive pulse 
pressures — check for faulty pump tubes, absence of surge 
suppressors, or improperly made or placed suppressors; (2) air 
bubbles in flowcell — check for absence of debubbler bypass, 
blockage in reagent pump tube, or periodic emptying of sample 
trap (latter results if air flow to distn trap becomes too great); 
{3) excessive H 2 S0 4 carryover — check for too high temp, in oil 
bath, improper H 2 S0 4 concn, or too high vac. on system; {4) air 
flow imbalances — check flowmeter settings, trapped air in tub- 
ing, or leak or block in system; (5) high F content in samples 
(baseline may not return to normal between samples) — dil. or 
check sampling speed and sample-to-wash ratio. 

(b) Irregular peaks.— Asymmetrical or double peaks or peaks 
with shoulders may result from: (7) baseline irregularities; (2) 
interfering substances from sample or impure reagents; (3) 
inadequate buffer concn; or (4) excessive amts solids in distn 
coil. Presence or accumulation of solids may be due to insuffi- 
cient flow of H 2 S0 4 , too large sample, excess CaO or NaOH in 
sample, inadequate suspension of particles in samples, or lack 
of proper air segmentation in sample tubing. 

(c) Poor reproducibility. — Check for: improper sample pickup; 
faulty pump tubes; inadequate washing of distn coil between 
samples; large deviations in acid concn, temp., or air flow in 
distn coil; or changes in vac. on waste system. 

Selenium 

3.096 * Gravimetric Method (2fl) — Official Final Action ic 

(Applicable to materials contg >2 ppm Se) 
See 3.073, 11th ed. 

Fluorometric Method (29) — Official Final Action 
{Caution: See 51.008, 51.019, 51.026, and 51.028.) 



3.097 



Apparatus 



(a) Micro-Kjeldahl flasks.— -30 mL Pyrex, ca 170 mm total 
length with ? 12/18 outer joint at mouth. 

(b) Air condensers. — 10 x 140 mm Pyrex tubes with f 12/18 
inner joint. 

(c) Micro-Kjeldahl digestion unit with glass fume duct. — Fit 
rack to hold flasks and attached air condensers in nearly upright 
position during early stages of digestion. Use in fume hood. 

(d) Fluorometer. — Capable of illuminating sample at 369 nm 
and measuring fluoresced light at 525 nm. Turner Associates, 
2524 Pulgas Ave, Palo Alto, CA 94303, Model 110 or 111 filter 
fluorometer equipped with std lamp No. 110-850, primary filter 
No. 7-60, and secondary filter No. 58 is satisfactory. Model 430 
spectrofluorometer set to above wavelengths is also satisfactory. 

3.098 Reagents 

(Use deionized H 2 distd in glass for prepg solns and dilns.) 

(a) Nitric acid.— Redistd in glass. 

(b) Hydroxylamine-ethyienediaminetetraacetic acid soln. — 
Add ca 20 mL H 2 to 1.9 g EDTA (acid form). Slowly add ca 5/V 
NH 4 OH with stirring until EDTA just dissolves. Some excess 

* Surplus method — see inside front cover. 



NH4OH is not harmful. Dissolve 6 g NH 2 OH.HCI in 100 mL H 2 0. 
Combine solns and dil. to 250 mL with H 2 0. 

(c) Cresol red indicator. — Dissolve 0.1 g cresol red in 10 mL 
H 2 and 1 drop 50% NaOH soln. Dil. to 50 mL with H 2 0. 

(d) Selenium std soln.— 0.3 fig Se/mL Add 10 mL HN0 3 to 
30.0 mg Se (purity ^99%) and warm to dissolve. Dil. to 100 mL 
with H 2 0, mix well, and transfer exactly 1 mL to micro-Kjeldahl 
flask. Add 2 mL 70% HCI0 4 and 1 glass bead. Boil gently to 
HCIO4 fumes and cool. Add 1 mL H 2 and 1 mL HCI (1 +4); heat 
30 min in boiling H 2 bath. Transfer to 1 L vol. flask and dil. to 
vol. with ca 1/V HCI. Store in all-glass container. Soln is stable 
several months at room temp. 

(e) Decalin. — Eastman Kodak No. 1905 decahydronaphthal- 
ene, or equiv. 

(f) 2,3-Diaminonaphthalene {DAN) soln. — Prep, soln in semi- 
darkened room or in room with only yellow light at time of 
detn. Protect from light and prep, fresh for each set of detns. 
Add 50 mL ca 0.1/V HCI to 0.05 g DAN (available from ICN-K&K 
Laboratories, Inc.). Place in 50° H 2 bath in dark 15 min. Cool to 
approx. room temp, and ext twice with 10 mL decalin, shaking 
vigorously each time and discarding decalin. Filter thru paper 
satd with H 2 0. For >8 detns, prep, larger amt. 

3.099 Preparation of Samples 

Grind air-dried samples to pass No. 18 or finer sieve. Cut fresh 
or wet samples finely with scissors or knife, or grind in food 
chopper to assure representative sample. 

Some plants {e.g., Astragalus bisulcatus, A. racemosus, Stan- 
leya bipinnata, and Oonopsis condensata) contain Se in volatile 
form that is lost during drying. Analyze these plants without 
drying. With usual agricultural crops, this is not a problem if 
drying is performed at 60-70°. 



3.100 



Preparation of Fluorometric Blanks 
and Standard 



(a) Blank. — Place 1 mL H 2 in micro-Kjeldahl flask. (For sam- 
ples contg <0.1 ppm, carry 10 mL HN0 3 as blank thru entire 
detn.) 

(b) Std. — Place 1.0 mL std Se soln in micro-Kjeldahl flask. 
Add 2 mL 70% HCI0 4 to each flask and continue as in detn, 

beginning "Mix contents of flasks . . ." 



3.101 



Determination 



(a) Samples containing 4 or more ppm selenium. — Weigh 
=£1 g sample (air-dried wt basis) contg =s0.4 ^g Se into micro- 
Kjeldahl flask. Add 1 glass bead, previously cleaned with HN0 3 . 
Add 10 mL HN0 3 and let stand at room temp. ^4 hr. (Use 5 mL 
HNO3 for samples <0.5 g.) Affix air condenser and place flask 
in nearly upright position on micro-Kjeldahl digestion unit. Heat 
ca 15 min with low flame and then increase heat until HN0 3 
condenses in lower part of condenser. Heat 10 min longer, turn 
off burner, and let cool 5 min. Wash down sides of flask with 2 
mL 70% HCIO4 thru air condenser. Swirl flask and continue 
refluxing 15 min. Remove condenser and continue heating, 
drawing off fumes in fume duct, until HCI0 4 fumes appear and 
then 15 min longer. Cool, add 1 mL H 2 0, and again heat to 
HCIO4 fumes and 1-2 min longer. Cool, and add 1 mL H 2 0. 

Mix contents of flasks and add 1 mL HCI (1+4) to each. Place 
in boiling H 2 bath 30 min. Cool to ca room temp. 

To each flask add 5 mL NH 2 0H-EDTA soln and 2 drops cresol 
red indicator. Neutze to yellow with ca 5/V NH 4 OH and add HCI 
(1 +4) to orange-pink. From this point, perform all operations in 
semidarkened room or room with yellow light only. Prep. DAN 
soln, add 5 mL to each flask, and dil. to neck with ca 0.1/V HCI. 
Mix and place in 50° H 2 bath in dark 25 min. 



46 



3. Plants 



AOAC Methods (1980) 



Remove flasks from H 2 bath and cool to ca room temp, in 
pan of H 2 0. Pour solns into 125 mL separators with Teflon 
stopcocks and contg 10.0 mL decalin. Shake vigorously >30 sec, 
let stand ca 1 min, and drain and discard lower layer. Wash 
decalin twice by shaking vigorously ^15 sec with 25 mL ca 0.1/V 
HCI. (VirTis, Rt 208, Gardiner, NY 12525, Extractomatic shaker 
with 100 mL separators may be substituted. When used, shake 
ext 5 min and wash 1 min periods.) Transfer decalin layer to 12 
mL centrf. tubes and centrf. 2 min at moderate speed. Pour 
decalin soln into fluorometer tubes, zero fluorometer against 
decalin, and read all tubes at 525 nm within 5 min. Correct std 
and unknown readings for blank, 

ppm Se = 0.3 x sample reading/std reading x g sample. 

(b) Samples containing less than 4 ppm selenium. — Proceed 
as in (a) thru second par. Dil. digest to adequate vol. and take 
aliquot contg ca 0.3 ^g Se for detn. Alternatively, digest sample 
in 10 vols HN0 3 2 hr on steam bath. Dil. to definite vol., and 
carry appropriate aliquot thru detn. Latter method is especially 
applicable when proper sampling requires large sample. Do not 
dil. decalin soln contg piazselenol, as this introduces errors. 



Boron {30) — Official Final Action 
Quinalizarin Method 



3.102 



Reagents 



(a) Dilute sulfuric acid.— 0.36/V. Dil. 10 mL H 2 S0 4 to 1 L. 

(b) Calcium hydroxide saturated soln. —Filter before use. 

(c) Quinalizarin soln.— Dissolve 45 mg quinalizarin in 1 L 
95-96% H 2 S0 4 . 

(d) Boron std soln.— 0.5 mg B/mL Dissolve 2.860 g H 3 B0 3 
and dil. to 1 L with H 2 0. Prep, working stds by further diln with 
H 2 0. 



3.103 



Determination 



Place 1 .00-2.00 g dry, ground plant material in Pt or Si0 2 dish. 
Add 5 mL satd Ca(OH) 2 soln and dry at 105°. Carefully volatilize 
over burner, ash in furnace 1 hr at 600°, and cool. Add exactly 
1 or 1 5 mL 0.36/V H 2 S0 4 , break up ash with glass rod, stir gently, 
and filter. Transfer 2 mL filtrate to colorimeter tube, add an exact 
amt (e.g., 15 mL) quinalizarin reagent, stopper, and mix by 
swirling gently. Let stand at room temp. 24 hr (or until both 
unknowns and stds have cooled to same temp.). Shake tube 
again immediately before reading in photoelec. colorimeter (620 
nm filter). 

Adjust colorimeter to 100% T with blank soln prepd as above 
but using 2 mL H 2 in place of sample soln. Prep, std curve with 
series of stds contg 0.5-10 jjlq B/mL. 



OTHER CONSTITUENTS 

Sugars (37) — Official Final Action 

3.104 Preparation of Solution 

(a) General method. — Prep, fresh sample as in 3.002(b). Pour 
ale. soln thru filter paper or extn thimble, catching filtrate in vol. 
flask. Transfer insol. material to beaker, cover with 80% alcohol, 
warm on steam bath 1 hr, let cool, and again pour ale. soln thru 
same filter. If second filtrate is highly colored, repeat extn. 
Transfer residue to filter, let drain, and dry. Grind residue so 
that all particles will pass thru 1 mm sieve, transfer to extn 
thimble, and ext 12 hr in Soxhlet app. with 80% alcohol. Dry 
residue and save for starch detn. Combine ale. filtrates and dil. 
to vol. at definite temp, with 80% alcohol. 

For dried materials, grind samples finely, and mix well. Weigh 



sample into beaker, and continue as above, beginning ". . . 
cover with 80% alcohol, . . ." 

(b) Applicable when starch is not to be determined. — Prep, 
fresh sample as in 3.002(b), but boil on steam bath 1 hr. Decant 
soln into vol. flask, and comminute solids in high-speed blender 
with 80% alcohol. Boil blended material on steam bath 0.5 hr, 
cool, transfer to vol. flask, dil. to mark with 80% alcohol at room 
temp., filter, and take aliquot for analysis. 

Grind dry material to pass No. 20 sieve or finer, transfer 
weighed sample to vol. flask, and add 80% alcohol and enough 
CaC0 3 to neutze any acidity. Boil 1 hr on steam bath, cool, adjust 
vol. at room temp, with 80% alcohol, filter, and take aliquot for 
analysis. 



3.105 



Clarification with Lead 



Place aliquot ale. ext in beaker on steam bath and evap. off 
alcohol. Avoid evapn to dryness by adding H 2 if necessary. 
When odor of alcohol disappears, add ca 100 mL H 2 and heat 
to 80° to soften gummy ppts and break up insol. masses. Cool 
to room temp, and proceed as in (a) or (b): 

(a) Transfer soln to vol. flask, rinse beaker thoroly with H 2 0, 
and add rinsings to flask. Add enough satd neut. Pb{OAc) 2 soln 
to produce flocculent ppt, shake thoroly, and let stand 15 min. 
Test supernate with few drops of the Pb(OAc) 2 soln. If more ppt 
forms, shake and let stand again; if no further ppt forms, dil. to 
vol. with H 2 0, mix thoroly, and filter thru dry paper. Add enough 
solid Na oxalate to filtrate to ppt all the Pb, and refilter thru dry 
paper. Test filtrate for presence of Pb with little solid Na oxalate. 

(b) Add twice min. amt of satd neut. Pb(OAc) 2 soln required 
to cause complete pptn, as found by testing portion of supernate 
with few drops dil. Na oxalate soln. Let mtxt. stand only few 
min; then filter into beaker contg estd excess of Na oxalate 
crystals. Let Pb ppt drain on filter and wash with cold H 2 until 
filtrate no longer gives ppt in oxalate soln. Assure excess of 
oxalate by testing with 1 drop Pb(OAc) 2 . Filter and wash pptd 
Pb oxalate, catching filtrate and washings in vol. flask. Dil. to 
vol. with H 2 and mix. 

3.106 Clarification with Ion-Exchange Resins [32) 

Place aliquot ale. ext, 3.104, in beaker and heat on steam bath 
to evap. alcohol. Avoid evapn to dryness by adding H 2 0. When 
odor of alcohol disappears, add ca 15-25 mL H 2 and heat to 
80° to soften gummy ppts and break up insol. masses. Cool to 
room temp. Prep, thin mat of Celite on filter paper in buchner 
or on fritted glass filter and wash until H 2 comes thru clear. 
Filter sample thru Celite mat, wash mat with H 2 0, dil. filtrate 
and washings to appropriate vol. in vol. flask, and mix well. 

Place 50.0 mL aliquot in 250 mL erlenmeyer; add 2 gAmberlite 
IR-120{H) analytical grade cation (replaced by REXYN 101(H) 
resin, Fisher Scientific Co.} and 3 g Duolite A-4{0H) anion ion 
exchange resins. Let stand 2 hr with occasional swirling. Take 
5 mL aliquot deionized soln and det. reducing sugars as glucose 
as in 31.053. 



3.107 
See 31.053. 



Glucose 
Micro Method— Official Final Action 



Fructose [33] — Official Final Action 

3.108 Reagents 

(a) Glucose oxidase preparation. — Add slowly, stirring con- 
stantly, 100 mL H 2 to 5 g glucose oxidase prepn ("DeeO L- 
750" code 4633000, Miles Laboratories, Inc., 1127 Myrtle St, 



AOAC Methods (1980) 



Starch 



47 



Elkhart, IN 46514). Stir ca 1 min and centrf. or filter to obtain 
clear soln. Add ca 1 mL CHCI 3 and refrigerate. Soln is stable 3*1 
month. 

(b) Mcllvaine's citrate-phosphate buffer. — Dissolve 214.902 g 
Na 2 HP0 4 .12H 2 and 42.020 g citric acid in H 2 and dil. to 1 L 



3.109 



Determination 



To suitable aliquot add !4 its vol. of buffer to give pH ca 5.8. 
Add 30% as much glucose oxidase prepn as estd glucose 
content (for 500 mg glucose add 150 mg glucose oxidase, i.e., 
3 mL soln), and few drops 30% H 2 2 (omit if Somogyi method 
is to be used in detn). Let stand overnight at room temp. 

Det. fructose by Somogyi micro method, 31.053, or by Munson- 
Walker method, 31.038-31.039, using Table 3:02. Check equivs 
in range of interest, using pure fructose as std, and correct as 
necessary. 

Table 3:02 Abbreviated Munson and Walker Table for 
Calculating Fructose 

{From Officiai and Tentative Methods of Analysis, 
AOAC, 5th Ed., 1940) 



Cuprous Oxide 




Cupr 


ous Oxide 




mg 


Fructose mg 




mg 


Fructose mg 


10 


4.5 




300 


148.6 


50 


23.5 




350 


174.9 


100 


47.7 




400 


201.8 


150 


72.2 




450 


229.2 


200 


97.2 




490 


253.9 


250 


122.7 




— 


— 



Reducing Sugars — Official Final Action 

3.110 Munson- Waiker Genera/ Method 
See 31.038. 

3.111 * Quisumbing-Thomas Method * 
See 31.048-31.049,1 1th ed. 

Sucrose — Official Final Action 

3.1 12 Hydrochloric Acid Inversion 

Using aliquot of cleared soln, 3.105, proceed as in 7.079. 

3.113 Invertase Inversion 

(a) For piants giving hydrolysis end point within 2 hours.-^ 
Pipet aliquot of cleared soln, 3.105, into 400 mL Pyrex beaker 
and make slightly acid to Me red with HOAc. Add 3 drops 1% 
soln of Wallerstein invertase scales. Let mixt. stand at room 
temp. 2 hr. Add reagents as in 31.035, and det. reducing power. 
Calc. results as invert sugar. Deduct reducing power of original 
soln, also expressed as invert sugar, and multiply difference by 
0,95. 

(b) For plants giving slower hydrolysis end point. — Place 
aliquot of soln, 3.105, in small vol. flask. Make slightly acid to 
Me red with HOAc. Add 3 drops 1 % soln of Wallerstein invertase 
scales and few drops toluene. Stopper flask and let stand 
overnight or longer at room temp. Dil. to vol. with H 2 and use 
aliquot for reducing power as above. 

3.114 Ether Extract — Official Final Action 
See 7.056. 

3.115 Crude Fiber — Official Final Action 
See 7.061-7.065. 



Total Nitrogen (Crude Protein) — Official Final Action 

3.116 Kjeldahl Method for Nitrate-free Samples 
See 2.057. 

3.117 Kjeldahl Method for Nitrate-Containing Samples 
See 2.058. 

3.118 Automated Method 
See 7.021-7.024. 

3.119 Semiautomated Method 
See 7.025-7.032. 



Starch {34) — Official Final Action 



3.120 



Reagents 



(a) Iodine-potassium iodide soln. — Grind 7.5gland7.5gKI 
with 150 mL H 2 0, dil. to 250 mL, and filter. 

(b) Alcoholic sodium chloride soln. — Mix 350 mL alcohol, 80 
mL H 2 0, and 50 mL 20% NaCI soln, and dil. to 500 mL with H 2 0. 

(c) Alcoholic sodium hydroxide soln. — 0.25/V. Mix 350 mL 
alcohol, 100 mL H 2 0, and 25 mL 5/V NaOH, and dil. to 500 mL 
with H 2 0. 

(d) Dilute hydrochloric acid.—OJN. Dil. 60 mL HCI to 1 L with 
H 2 0. 

(e) Somogyi phosphate sugar reagent. — Dissolve 56 g an- 
hyd. Na 2 HP0 4 and 80 g Rocheile salt in ca 1 L H 2 0, and add 200 
mL 1.00/V NaOH. Then slowly add, with stirring, 160 mL 10% 
CuS0 4 .5H 2 soln. Dissolve 360 g anhyd. Na 2 S0 4 in this soln, 
transfer to 2 L vol. flask, and add exactly 200 mL 0.1/V KI0 3 soln 
(3.5667 g/L). Dil. to vol., mix well, let stand several days, and 
filter thru dry paper into dry flask, discarding first 50 mL filtrate. 
Store reagent at 20-25°. tt is 0.01/V with respect to KI0 3 ; 5.00 
mL is equiv. to 10 mL 0.005/V Na 2 S 2 3 . 

Det. glucose factor of reagent as follows: Accurately weigh 
150 mg NBS glucose SRM into 1 L vol. flask, dissolve in H 2 0, dil. 
to vol., and mix well. Transfer 5 mL aliquot to 25 x 200 mm 
Pyrex test tube, add exactly 5 mL Somogyi reagent, stopper with 
size 00 crucible, and heat (together with several blanks contg 5 
mL H 2 and 5 mL reagent) exactly 15 min in boiling H 2 bath. 
Titr. as in detn. From difference between blank and std titrns, 
calc. mg glucose equiv. to 1 mL exactly 0.005/V Na 2 S 2 3 . Effective 
range for detn is 0.05-1.0 mg glucose in 5 mL aliquot. 

(f) Sodium thiosuifate std soln.— 0.005/V. Dissolve 2.73 g 
Na 2 S 2 3 .5H 2 in H 2 and dil. to 2 L Stdze daily as follows: Add 
1 mL Kl soln, (g), and 3 mL 1.5/V H 2 S0 4 to 5 mL Somogyi sugar 
reagent. Let stand 5 min, and titr. with Na 2 S 2 3 soln, adding 
starch indicator, (h), just before end point. 

(g) Potassium iodide soln. — 2.5%. Stabilize with little Na 2 C0 3 . 
(h) Starch indicator. — Make 1.5 g sol. starch into paste with 

few mL H 2 0, and add slowly, with stirring, to 300 mL boiling 
H 2 0. 



3.121 



Determination 



Select sample as in 3.001, remove all foreign matter, dry, and 
grind to pass No. 80 sieve. Accurately weigh 0.1-1.0 g powd 
sample contg ca 20 mg starch into 25 x 150 mm Pyrex test 
tube. Add ca 200 mg fine sand and 5 mL H 2 0, and mix well with 
stirring rod to wet sample. Heat tube in boiling H 2 bath 15 min 
to gelatinize starch. Cool to room temp., and place in 22-25° 
bath. Add 5 mL 60% HCI0 4 rapidly with const agitation. Grind 
tissue against lower wall of tube with stirring rod for approx. 

* Surplus method—see inside front cover. 



48 



3. Plants 



AOAC Methods (1980) 



min at time. Repeat grinding frequently during 30 min; then 
without delay, transfer quant, to 100 ml_ vol. flask with H 2 0. Add 
3 mL 5% uranyl acetate soln to ppt protein, dil. to vol. with H 2 0, 
mix well, and centrf. portion of mixt. Pipet 10 mL clear supernate 
into 25 x 150 mm test tube. Add ca 100 mg Celite, 5 mL 20% 
NaCI soln, and 2 mL i-KI reagent, and mix well. Let stand 
overnight, centrf., and decant. 

Wash starch-l ppt by suspending it in 5 mL ale. NaCI soln, 
centrf., and decant. Add 2 mL ale. NaOH soln to packed ppt. 
Gently shake and tap tube until ppt is no longer blue. (Do not 
use stirring rod; allow ample time for complex to decompose.) 
Wash walls of tube with 5 mL ale. NaCt soln, centrf. liberated 
starch, and wash with 5 mL ale. NaCI soln as before. Add 2 mL 
0.7/V HCI to ppt. Stopper tube loosely with size 00 crucible, and 
heat 2.5 hr in boiling H 2 bath. (Bath should have cover with 
holes to accommodate tubes; holes not occupied by tubes must 
be covered.) Cool, and transfer quant, to 25 mL vol. flask. Add 
drop phenol red, 50.008, and neutze with 1/V NaOH. Discharge 
color with 0. 1N oxalic acid, dil. to vol., and mix well. Transfer 
5 mL aliquot to 25 x 200 mL Pyrex test tube, add exactly 5 mL 
Somogyi reagent, and stopper tube with size 00 crucible. Heat 
together with several blanks contg 5 mL H 2 and 5 mL Somogyi 
reagent in vigorously boiling H 2 bath exactly 15 min. Remove 
tube from bath and cool to 25-30°. Add 1 mL 2.5% Kl soln down 
wall of tube without agitation and then add 3 mL 1.5/V H 2 S0 4 
rapidly with agitation. After all Cu 2 dissolves, titr. soln with 
0.005/V Na 2 S 2 3 , adding starch indicator, (h), just before end 
point is reached. Treat blank solns similarly. 

% Starch = [50(mL blank - mL sample) 

x 0.90/mg sample] x (/V/0.005) x G x 100, 

where 50 = diln factor, 0.90 = factor glucose to starch, N - 
actual normality Na 2 S 2 3 soln, and G - mg glucose equiv. to 1 
mL 0.005/V Na 2 S 2 3 . 

Lignin (35) — Official Final Action 
Direct Method 



3.122 



Preparation of Sample 



benzene soln (32 parts alcohol and 68 parts benzene by wt). Dry 
material in oven to free it from solvs and place in flask of 
suitable size. Add 150 mL H 2 0/g sample, and reflux 3 hr. Filter 
mixt. while still hot, preferably thru weighed fritted glass cru- 
cible, and transfer extd material to flask. Add 1% HCI (111 g 
coned HCI + 3890 mL H 2 0) in proportion of 150 mL acid soln/g 
plant material, and reflux 3 hr. Filter mixt. while still hot thru 
fritted glass crucible previously used, wash with H 2 until acid- 
free, dry at 105°, and weigh. Calc. % total loss due to successive 
extn with alcohol-benzene soln, hot H 2 0, and 1% HCI. (With 
samples not especially rich in carbohydrates and proteins, extn 
with hot H 2 may be omitted.) 

3.123 Apparatus 

App., Fig. 3:06, consists of: (1) 1.5 L bottle, A, to which is 
attached by 2-hole rubber stopper 250 mL dropping funnel, C, 
having lower end of stem bent as illustrated and placed close 
to bottom of A; (2) Drechsel gas-washing bottle, D; (3) 3 Pyrex 
test tubes, 38 x 300 mm diam., G, G r , G" f connected in parallel 
by device, 0, and immersed in wooden box, L, filled with 
crushed ice, H; and (4) bottle contg H 2 for absorption of excess 
HCI, K. G, G' f and G" are provided with 2-hole rubber stoppers; 
glass tube with right angle bend extends thru 1 hole nearly to 
bottom of test tube, and similar tube extending ca 10 mm into 
test tube passes thru other hole. Rubber connections and 
stopcocks for regulating flow of gas are provided as indicated 
in diagram. A is filled with ca 500 mL H 2 S0 4 and C with HCI; HCI 
flowing thru stoptockfi into A generates HCI gas, which is dried 
by H 2 S0 4 in D, and flows into G, G' , and G" contg samples and 
fuming HCI reagent. 



3.124 



Reagent 

{Caution: See 51.031.) Density 



Grind sample in mill to pass No. 80 sieve and dry at 105°. Ext 
weighed sample (5-10 g) 30 hr in Soxhlet app. with alcohol- 



Fuming hydrochloric acid. 
1.212-1.223 at 15°. To 500 g NaCI in 1 L g-s Pyrex distg flask, 
add cold soln of 250 mL H 2 in 450 mL H 2 S0 4 . Connect side tube 
of distg flask to glass tube passing thru H 2 S0 4 wash bottle, and 
connect outlet tube of H 2 S0 4 wash bottle to another glass tube, 
immersed in flask contg 3 L HCI. Surround flask contg HCI with 
crushed ice. Heat distg flask with small flame and pass HCI gas 
into acid soln until it attains sp gr of 1.212-1.223 at 15°. Keep 




RG. 3:06 — Apparatus for determining Signin 



AOAC Methods (1980) 



Pigments 



49 



reagent refrigerated at =£0°. If only few detns are to be made, 
prep, correspondingly smaller ami 



3.125 



Determination 



Weigh three 1 g samples of extd and dried sample in weighing 
bottle and place in 3 large test tubes, G, G', and G". Add 20 mL 
of the reagent to each tube, using this acid to wash down any 
particles clinging to sides. When all material is wet with reagent 
add addnl 30 mL reagent. Add ca 3 drops capryl alcohol to 
minimize foaming. Place test tubes, G, G', and G", in wooden 
box, L, and surround with crushed ice. Lubricate tubes F, F', and 
F" with drop of glycerol so that they move easily thru holes in 
rubber stoppers. Lead dry HCI gas from generator into reaction 
mixts thru tubes F, F' , and F" (f and F" are shown in top view), 
which reach nearly to bottom of tubes G, G' , and G". Regulate 
flow of gas thru reaction mixts in G, G' , and G" by stopcocks 
shown in top view, continuing passage of gas 2 hr. {At first 
rather slow stream of gas passes in, but during last 1 5 min, flow 
is fairly rapid.) 

After reaction period, discontinue flow of gas, and disconnect 
long tubes F, F' , and F" and outlet tubes of test tubes G, G', and 
G" from O and P. Pull tubes F, F' , and F" just above surface of 
reaction mixt., and close with short pieces of rubber tubing 
having one end plugged with short piece of glass rod. Similarly 
close off outlet tubes, N, AT, and N". Place tubes contg reaction 
mixt. in cold room or refrigerator (8-10°) 24 hr. 

Transfer contents of G, G', and G" to 1 L erlenmeyers, taking 
care to remove any material adhering either to inside or outside 
of tubes F f F', and F". Dil. reaction mixts to 500 mL with H 2 0. 
Connect flasks to reflux condensers and boil 1 hr. Prep. 3 
gooches in usual manner, dry at 105°, and weigh. Ignite one of 
weighed crucibles, X, over Bunsen burner, cool in desiccator, 
and reweigh. Let contents of flasks cool to room temp, and filter 
thru weighed gooches. Wash ppts collected in gooches with hot 
H 2 0, dry at 105°, and weigh in weighing bottles. Ignite crude 
lignin in crucible X over Bunsen flame and det. wt ash. Place 
one of other 2 gooches in wide-neck Kjeldahl flask and det. % 
N in crude lignin as in 2.057. If methoxyl in lignin is to be detd, 
collect pptfrom one of flasks in dried (105°) fritted glass crucible 
and proceed as in 47.050. 

Wt lignin = wt crude lignin - wt ash - wt crude 
protein (N x 6.25). 

Calc. % lignin in original dry unextd material. 

3.126 indirect Method {36) 

{Caution: See 51.086.) 

Ext 1 g sample with alcohol-benzene (1+2) 4 hr in Soxhlet or 
comparable app. (extn vessel may be either coarse porosity 
Alundum or paper thimble, closed at top with filter paper or 
plug of cotton). Wash sample in thimble with suction, using 2 
small portions alcohol followed by 2 small portions ether. Heat 
at 45° in nonsparking oven to drive off ether, and transfer sample 
to 250 mL wide-mouth erlenmeyer. Add 40 mL 1% soln of 
pepsin in 0. 1N HCI, wetting sample well by adding small portion 
reagent, stirring or shaking thoroly, and finally washing down 
sides of flask with remaining soln. Incubate at 40° overnight. 

Add 20-30 mL hot H 2 and filter, using filter stick. (Filter sticks 
are made with Pyrex fritted glass disk, 30 mm diam., medium 
porosity. Thin layer of pre-ashed diat. earth (Hyflo Super-Cel, or 
similar filter-aid) is sucked onto disk from H 2 suspension. This 
is usually enough for easy filtration; if not, add extra Super-Cel 
to material being filtered. Some sticks filter slowly with some 
samples. It is advisable to obtain more than needed and discard 
slow-filtering ones. It is convenient to arrange filter sticks in set 
of 12 attached to vac. manifold by rubber tubing.) 



Repeat washing twice and then wash residue into flask by 
forcing 7-8 mL 5% (w/w) H 2 S0 4 downward thru filter stick, using 
air pressure. Wash stick further with the H 2 S0 4 , finally adding 
enough to bring total vol. to ca 150 mL. Reflux vigorously on 
hot plate 1 hr, adding H 2 occasionally to maintain original vol. 
Filter off acid. Wash residue with three 20-30 mL portions hot 
H 2 0, two 15-20 mL portions alcohol, and two 15 mL portions 
ether. Leave vac. on few min to dry residue, and transfer from 
stick to flask by tapping and brushing. Heat to drive off any 
residual ether. If disk formed upon drying is difficult to break up 
into finely divided state (sometimes in case of immature plant 
samples), disperse residue in ether in flask and then boil off 
ether on steam bath. Add 20 mL 72% (w/w) H 2 S0 4 at 20° to 
residue and hold 2 hr at 20°, stirring occasionally. Add 125 mL 
H 2 0, filter, wash once with 20 mL hot H 2 0, and filter again. Wash 
residue from filter stick and reflux as before 2 hr, using 150 mL 
3% (w/w) H 2 S0 4 . Filter residue onto gooch with asbestos pad 
and wash with hot H 2 until acid-free. Dry at 105-110° and det. 
lignin by loss in wt on ignition at 600°. 



PIGMENTS 

Chlorophyll — Official Final Action 

Photoeiectric Colorimetric Method for Total 
Chlorophyll Only {37) 

3.127 Apparatus 

(a) Mortar and pestle. — Deep glass mortar ca 10 cm id with 
well-defined lip. 

(b) Photoelectric colorimeter. — Calibrate for chlorophyll, us- 
ing plant ext as in 3.129 and light filters with max. T near 660 
nm. (Combination of Corning Glass Works filters CS No. 2-60 
and 1-58 (Glass No. 2408 and 3965, resp.) is suitable.) 

(c) Wash bottles. — Type fitted with rubber bulb, permitting 
operation with one hand. 

(d) High-speed blender. — Waring Blendor, or equiv. 



3.128 



Reagents 

(1) Undild acetone and (2) 85% aq. soln by vol. 



(a) Acetone. 
Com. acetone, tech. grade, is satisfactory. 

(b) Quartz sand. — Acid-washed and dried. 



3.129 



Determination 



{Caution: See 51.004, 51.040, and 51.046.) 



Select field material carefully to ensure representative sample. 
Remove representative portion from field sample, and if fresh, 
cut finely with hand shears and mix as thoroly as possible. Grind 
dried material in mill and mix thoroly. 

Weigh 1-5 g into mortar and add ca 0.1 g CaC0 3 or Na 2 C0 3 . 
Macerate tissue with pestle, add quartz sand, and grind short 
time; then add 85% acetone, little at time, and continue grinding 
until tissue is finely ground. Transfer mixt. to funnel, filter with 
suction, and wash residue with 85% acetone. Return residue to 
mortar with more 85% acetone and grind again. Filter and wash 
as before. Repeat procedure until tissue is devoid of any green, 
and washings are colorless. (It is advisable to grind residue at 
least once with undild acetone and then to add enough H 2 at 
end to bring acetone concn to 85%. High-speed blender may be 
used instead of mortar to macerate and ext tissue {see 3.132), 
but each investigator should satisfy himself that device used 
exts tissue completely.) When extn is complete, transfer filtered 
ext to vol. flask of appropriate size and dil. to vol. 

Measure T of soln with photoelec. colorimeter, and read amt 
of chlorophyll present from curve relating 7" and concn. Express 



50 



3. Plants 



AOAC Methods (1980) 



chlorophyll values as mg/g tissue, or in other convenient 
manner. 

Calibrate photoelec. colorimeter as follows: Ext sample of 
fresh, green leaf material with 85% acetone, filter, wash residue, 
and dil. ext to vol. as above. Make series of dilns of ext and 
measure T of original and of each of dild solns with instrument 
in same manner as when chlorophyll prepn is being used as 
calibration std. Transfer aliquot of original ext to ether and 
evaluate total chtorophyll spectrophtric as in 3.132(b) and (c). 
From value thus obtained, calc. chlorophyll content of original 
ext and that of each of dild solns, and construct curve relating 
concn of chlorophyll with T or A. 

Spectrophotometry Method for Total 

Chlorophyll and the a and b 

Components {38, 39) 

3.130 Apparatus 

Use app. in 3.127 (except photoelec. colorimeter), plus follow- 
ing: 

(a) Scrubbing tubes for washing ether solns, — Open tubes ca 
20 mm diam. to one end of each of which is sealed tube of 
smaller diam. drawn to fine jet at lower end. 

(b) Spectrophotometer. — Capable of isolating spectral region 
of ca 3 nm near 660 nm with negligible stray radiation. Tubulated 
cells with tightly fitting glass stoppers are recommended for 
work with ether. 

3.131 Reagents 

Those listed in 3.128 and following: 

Ether. — Com. grade is satisfactory without further purification. 



3.132 



Determination 



(Wash glassware with coned Na 3 P0 4 soln to remove traces of 
acid that may decompose chlorophyll.) 

(a) Extraction of chlorophyll from tissue. — Select and prep, 
sample as in 3.129. Disintegrate weighed portion (2-10 g, 
depending on chlorophyll content) of fresh plant tissue in 
blender cup that contains ca 0.1 g CaC0 3 , or by use of mortar 
as in 3.129. After tissue is thoroly disintegrated, filter ext thru 
buchner fitted with quant, paper. Wash residue with 85% 
acetone, 3.128(a), and if necessary, use little ether to remove 
last traces of pigment. If extn is incomplete, return residue and 
paper to blender container with more 85% acetone and repeat 
extn. Filter and wash, as before, into flask contg first filtrate. 
Transfer filtrate to vol. flask of appropriate size and dil. to vol. 
with 85% acetone. 

Pipet aliquot of 25-50 mL into separator contg ca 50 mL ether. 
Add H 2 carefully until it is apparent that ail fat-sol. pigments 
have entered ether layer. Drain and discard H 2 layer. Place 
separator contg ether soln in upper rack of support. Add ca 100 
mL H 2 to second separator placed in rack below first. Set 
scrubbing tube in place, and let ether soln run thru it to bottom 
of lower separator and rise in small droplets thru the H 2 0. When 
all soln has left upper separator, rinse it and scrubbing tube 
with little ether added from medicine dropper. Place scrubbing 
tube in upper separator and exchange its place in support with 
separator now contg ether soln. Drain and discard H 2 in upper 
separator, add similar portion of fresh H 2 to lower separator, 
and repeat washing process. Continue washing ether soln until 
all acetone is removed (5-10 washings). Then transfer ether soln 
to 100 mL vol. flask, dil. to vol., and mix. 

(b) Spectrophotometric measurements. — Add ca teaspoon- 
ful (ca 5 mL) anhyd. Na 2 S0 4 to 60 mL reagent bottle, and fill it 
with ether soln of pigment. When this soln is optically clear, 



pipet aliquot into another dry bottle and dil. with enough dry 
ether to give /I value of 0.2-0.8 at wavelength to be used. (Most 
favorable value is near 0.6 at 660 nm, since such soln yields 
satisfactory value at 642.5 nm.) 

Fill 2 clean g-s absorption ceils with dry ether from pipet and 
polish outside surfaces of each, first with cotton wet with alcohol 
and then with dry cotton. Place cells in instrument, and det. 
whether each gives same galvanometer deflection. If not, clean 
again or select cells that do, and do this daily. Empty one cell, 
fill it with the dried ether soln, and place in instrument. Adjust 
entrance and exit slits until spectral region isolated is 3-^4 nm 
at 660.0 nm. 

Det. whether instrument is in proper adjustment for wave- 
length by taking A readings thru soln against solv. at 1 nm 
intervals from 658 to 665 nm. Highest value should be at 660.0 
nm; if not, adjust instrument until it is, or make 660.0 nm 
readings at wavelength setting that gave highest A. With grating 
instrument, apply same correction at 642.5 nm; however, with 
prism instrument, correction at 642.5 nm must be obtained from 
wavelength calibration curve for particular instrument in use. 
Calibrate instrument for wavelength in this way often enough 
to ensure that it remains in proper adjustment. Det A at 660.0 
and 642.5 nm (or corrected settings) for each unknown soln. 

(c) Calculation of chlorophyll concentration.— Calc. total 
chlorophyll and each of a and b components (mg/L) as follows: 

(1) Total chlorophyll = 7.12y4 660 . + 16.8 /Ws- 

(2) Chlorophyll a = 9.93 ^ 6600 - 0.777 >4 642 . 5 . 

(3) Chlorophyll b = 17.6>4 642(5 - 2.81 /W . 



3.133 Supplementary Information 

Factors involved in spectrophtric analysis of chlorophyll sys- 
tem have been discussed in detail by Comar and Zscheile (39). 
These authors used Beer's law in form: 

c = (log 10 / //)/a x t [= A/a x t], 
where / is intensity of light transmitted by solv. -filled cell; / is 
intensity of light transmitted by soln-filled cell; c is concn of 
chlorophyll (g/L); a is absorptivity; t is thickness of soln layer 
in cm, and A is absorbance. 

Since, at given wavelength, observed A value of soln having 
2 components represents sum of A values of each of compo- 
nents, following equation holds in case of chlorophylls a and b 
at given wavelength: 

(4) >4 0bserved = A a + A b 

If 1 cm cell is used, this equation may be expressed as: 

(5) ^observed ~ d a C a + SftCft- 

Concns of chlorophylls a and b in given ether soln can now 
be calcd by equation (5) as follows: 

(a) Det. A for soln at 2 different wavelengths (660.0 and 642.5 
nm have been found advantageous for this purpose). 

(b) From table select proper absorptivities corresponding to 
wavelengths used. 

(c) Substitute observed/^ value and absorptivities in equation 

(5) for each of the 2 wavelengths used as illustrated for 660.0 
and 642.5 nm in equations (6) and (7). Solve these 2 equations 
simultaneously for 2 unknowns, the concns of chlorophylls a 
and/?. 

(6) v4 660 . = 102 c a + 4.50 c b . 

(7) >4 642 . 5 = 16.3 c a + 57.5 c b . 

Equations (1), (2), and (3) were derived this way. 

Criterion for accuracy of chlorophyll values detd by spectro- 
phtric method is agreement between analytical results detd 
from measurements at different wavelengths. Comar and 
Zscheile (39) demonstrated that measurements at 660.0 and 



AOAC Methods (1980) 



Tobacco 



51 



642.5 nm are convenient for routine analysis; however, readings 
may be made at other wavelengths to check these values. 
Absorptivities for chlorophylls a and b in ether soln that may be 
used for this purpose are presented in Table 3:03. 
These values may be used for calcns as follows: 

(a) Values for total chlorophyll and % composition may be 
calcd from A at 660.0 and 642.5 nm as described. 

(b) Check values for total chlorophyll may be calcd from A at 
intersection points 600.0, 581.0, and 568.0 nm. 

(c) Check values for % composition may be calcd from A for 
each of points 613.0 and 589.0 nm in combination with value of 
total concn obtained from (a) or (b). 

Table 3:03 Absorption constants used in analysis 
(after Comar and Zscheile [39)) 



3.139 



Determination 



Wavelength 




Absorptivities 
(for Ether Solns) 




nm 


Chlorophyll a Chlorophyll b 


660.0 


102 




4.50 


642.5 


16.3 




57.5 


600.0 


9.95 




9.95 


581.0 


8.05 




8.05 


568.0 


7.11 




7.11 


613.0 


15.6 




8.05 


589.0 


5.90 




10.3 



3.134 Carotenes — Official Final Action 
See 43.014-43.023. 



TOBACCO 

Moisture (40) — Official Final Action 

3.135 Apparatus 

(a) Drying oven. — Forced-draft, regulated to 99.5 ±0.5°. Sug- 
gested dimensions: 19 x 19 x 19" (48 cm). Approx. oven 
settings: fresh air intake vent Vs open; air control damper V* 
open; air exhaust vent Y 3 open. 

(b) Moisture dish. — Al, diam. 45-65 mm, depth 20-45 mm, 
with tight-fitting cover. 



3.136 



Determination 



Accurately weigh ca 5 g sample (ground to pass ^1 mm 
screen) into weighed moisture dish and place uncovered dish 
in oven. 

Do not exceed 1 sample/ 10 sq in. (650 sq cm) shelf space, 
and use only 1 shelf. Dry 3 hr at 99.5±0.5°. Remove from oven, 
cover, and cool in desiccator to room temp, {ca 30 min). Reweigh 
to nearest 1 mg and calc. % moisture. 



Chlorides (4?)— Official Final Action 
Potentio metric Method 

3.137 Reagent 

Silver nitrate std soln.— 0AN. Stdze against KCI as in detn. 

3.138 Apparatus 

(a) pH meter. — Leeds and Northrup, Sumneytown Pike, N 
Wales, PA 19454, Beckman Instruments, or equiv., equipped 
with Ag and glass electrodes. 

(b) Buret — 10 mL, graduated in 0.05 or 0.02 ml_, preferably 
reservoir-type. 



Accurately weigh ca 2 g sample, ground to pass No. 40 sieve, 
into 250 mL electrolytic beaker. Add 100 mL H 2 0, small amt at 
first to thoroly wet sample; then remainder. Let stand s=5 min 
at room temp., stirring intermittently. Pipet 5 mL HN0 3 (1+9) 
into mixt. and insert clean electrodes. Start mag. stirrer and 
continue stirring thruout titrn at rate that produces vigorous 
agitation without spattering. Titr. with std 0.1/V AgN0 3 soln to 
potential previously established as equivalence point. Det. equiv- 
alence point potential graphically by making several titrns on 
one or more tobacco samples. Recheck occasionally, and redet. 
when either electrode is replaced. Record vol. of titrant and 
calc: 

% CI = mL AgN0 3 x normality x 3.5453/g sample. 

Nitrogen (42) — Official Final Action 

Kjeldahl Method for Samples 
Containing Nitrates 

(For nitrate-free samples, omit salicylic acid 
and thiosulfate treatment.) 



3.140 



Reagents 



See 2.055 and the following: 

(a) Sodium hydroxide-thiosulfate soln. — Dissolve 500 g NaOH 
pellets and 40 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L 

(b) Indicators.— (1) Dissolve 1 g Me red in 200 mL alcohol; or 
(2) prep, mixed indicator by dissolving 0.8 g Me red and 0.2 g 
methylene blue in 500 mL alcohol. 



3.141 
See 2.056. 

3.142 



Apparatus 



Determination 

Place weighed sample (1-2 g) in digestion flask. Add vol. H 2 S0 4 
(contg 2 g salicylic acid/ 40 mL) corresponding to wt sample (35 
mL for 1 g, 40 mL for 2 g for N0 3 -contg samples; 20 and 25 mL, 
resp., for N0 3 -free samples). Shake until thoroly mixed; let stand 
3=30 min with occasional shaking; then add 5 g Na 2 S 2 3 .5H 2 0. 
Shake, let stand 5 min, and heat carefully until frothing ceases. 
Turn off heat, add 0.7 g HgO (or metallic Hg) and 1 5 g K 2 S0 4 , and 
boil briskly 1-1.5 hr after soln clears. 

Cool, add ca 200 mL H 2 0, cool to ca room temp., and add few 
Zn granules. Tilt flask and carefully add 50 mL NaOH-thiosulfate 
soln without agitation. Immediately connect flask to distn bulb 
on condenser whose tip is immersed in 50 mL std 0.1/V acid in 
receiving flask. Then rotate digestion flask carefully to mix 
contents. Heat until s*150 mL distillate collects, and titr. excess 
acid with std base, using Me red or mixed indicator. Correct for 
blank detn on reagents. 

Potassium (43) — Official Final Action 
3.143 Reagents 

(a) Potassium std soins. — (/) Stock soln.— 1000 ppm K. See 
3.020(a). (2) Working solns.— Place 0, 5, 10, 15, 20, 25, and 30 
mL stock soln in seven 1 L vol. flasks, add 40 mL 3/V HCI to each, 
and dil. to vol. with H 2 0. 

(b) Diatomaceous earth. — Celite 545, acid-washed. 



3.144 



(a) Flame photometer. 
quate for K analysis. 

(b) Chromatographic tube. 
disk. 



Apparatus 

Natural gas-air fuel, or equiv., ade 



-20 x 150 mm with coarse fritted 



52 



3. Plants 



AOAC Methods (1980) 



3.145 Preparation of Sample Solution 

Accurately weigh ca 0.5 g tobacco dust into ca 40 mL weighing 
dish. Add ca 1 g Celite and mix intimately with spatula. Transfer 
quant, thru powder funnel into chromatgctube. Add addnl Celite 
thru funnel into tube until 2.5 cm layer accumulates on top of 
sample-Celite mixt Compact sample and Celite by tapping tip 
of tube on table top, and insert tip of tube into neck of 1 L vol. 
flask. Add 40 mL 3/V HCI into tube by pipet or dispenser, washing 
down sides, and let elute into vol. flask. When liq. level reaches 
top of Celite, add 25 mL H 2 and let elute. Add second 25 mL 
portion H 2 0, let elute by gravity, or force thru rapidly with 
compressed air. Rinse tip of tube into vol. flask, dil. to vol. with 
H 2 0, and mix well. 



3.146 



Determination 



Det % T for sample eluate and K stds as specified in instruction 
manual of instrument. See also 3.023. 
Prep, calibration curve and det. ppm K of sample from curve. 
% K = ppm K x 0.1/g sample. 
% K 2 = ppm K x 0.1205/g sample. 

Glycerol, Propylene Glycol, and Triefhylene 

Glycol in Cased Cigarette Cut Filler and 
Ground Tobacco {44) — Official First Action 



{Caution: See 51.018 and 51.066.) 



3.147 



Apparatus 



(a) Gas chromatograph.—W\\\\ programmed temp, oven and 
W hot wire detector; F&M Model 720 (current models 5700 
series; Hewlett-Packard, Inc.), or equiv. Conditions: Detector 
bridge 140 ma; temps (°): injection 265, detector 280, column 
90-240 at 15°/min; He 60 mL/min adjusted, if necessary, to 
facilitate sepns; attenuation 4, adjusted according to sensitivity 
to yield peaks of sufficient size for accurate measurement (use 
same attenuation for all stds and samples); chart speed, 12"/hr. 

(b) Column.— 42 (105 cm) x 3 /i 6 " Cu tubing packed with 5% 
Carbowax 20M-terephthalic acid (TPA) on 60-80 mesh Chro- 
mosorb G AW-DMCS (Hewlett-Packard, Inc., No. 8501-6223 or 
Applied Science Laboratories, Inc., No. 04388). Prep, packing by 
placing 30.0 g Chromosorb in 500 mL $ r-b flask. Add soln of 
1.50 g Carbowax 20M-TPA in 150 mL CHCI 3 , and slurry. Remove 
CHCI 3 under vac. in rotary evaporator and air dry overnight at 
room temp. Condition new column 2 hr at 240°; then inject three 
30 ju,L samples tobacco ext before analyzing samples. Recon- 
dition columns removed from app. before use. 



3.148 



Reagents 



(a) Extracting soln. — Dil. 20.0 mL 1,3-butylene glycol stock std 
soln, (b), to 2 L with anhyd. MeOH. 

(b) 1,3-Butylene glycol stock std soln. — Accurately weigh 
20.00 g USP 1,3-butanediol into 100 mL vol. flask and dil. to vol. 
with anhyd. MeOH. 

(c) Glycerol stock std soln. — Accurately weigh 10.00 g USP 
glycerol into 100 mL vol. flask and dil. to vol. with extg soln. 

(d) Propylene glycol stock std soln. — Accurately weigh 5.00 
g USP propylene glycol into 100 mL vol. ffask and dil. to vol. with 
extg soln. 

(e) Tri ethylene glycol stock std soln. — Accurately weigh 5.00 
g triethylene glycol into 100 mL vol. flask and dil. to vol. with 
extg soln. 

(f) Humectant std solns. — Into each of four 100 mL vol. flasks, 
pipet 1.0, 2.0, 3.0, and 4.0 mL, resp., glycerol, propylene glycol, 
and triethylene glycol stock std solns. Dil. to vol. with extg soln. 
Each soln contains (in mg/100 mL): 





Propylene 




Triethylene 


Soln 


Glycol 


Glycerol 


Glycol 


1 


50 


100 


50 


2 


100 


200 


100 


3 


150 


300 


150 


4 


200 


400 


200 



3.149 



Extraction 



Place 10.00 g sample in 250 mL $ erlenmeyer. Pipet 100 mL 
extg soln into flask and stopper. Shake mech. 1 hr and let settle 
few min until supernate is clear. Alternatively, shake mech. 30 
min and let stand overnight. 



3.150 



Determination 



Prime column by injecting two 30 /xL aliquots supernate ext. 
Then alternately inject 30 fxL supernate exts and a humectant 
std soln until all samples and stds have been run, repeating ext 
injections, if necessary. (Sequence is ext^ ext u std u ext,, std 2 , 
ext 2 , std 3 , ext 3 , std 4 , ext 4/ std 1f ext 5 , std 2 , etc. If <4 exts are 
available, distribute ext injections among those available so that 
sequence thru std 4 is run.) Det. peak hts and calc. ratios of 
propylene glycol, glycerol, and triethylene glycol to butylene 
glycol for each std and sample soln. Plot peak ht ratios against 
polyol concn for std solns and construct std curve for each 
humectant. Det. concn in mg/100 mL for propylene glycol, 
glycerol, and triethylene glycol in sample soln from resp. std 
curves. 

% Humectant = (mg/100 mL) x 0.01. 

Total Alkaloids (As Nicotine) 

Distillation Method {45) — Official Final Action 
3.151 Apparatus 

(a) Distillation apparatus. —500 mL Kjeldahl flask fitted with 
inlet tube for steam, trap bulb, and condenser; Griffith still 
(Tobacco Sci. 1, 130(1957), available from Lab Glass, Inc., PO 
Box 5067, Kingsport, TN 37663); or other suitable steam distn 
app. 

(b) Spectrophotometer, — Beckman Instruments Model DU 
(replaced by Models 24/25) or other instrument capable of 
accurately measuring^ in 200-300 nm range, equipped with 1 
cm quartz cells. 



Reagents 

-Dissolve 300 g NaOH in 700 mL H 2 and 



3.152 

(a) Alkali-salt soln.- 
sat. with NaCI. 

(b) Silicotungstic acid soln {for gravimetric determination). — 
Dissolve 120 g Si0 2 .12W0 3 .26H 2 in H 2 and dil. to 1 L. (Soln 
should be clear and free from green color.) 

3 . 1 53 Standardization 

{Caution: Nicotine is very toxic. Avoid contact 
with skin and eyes. See 51.011 and 51.015.) 

Purify best grade of nicotine com. available by successive vac. 
distns until center cuts from 2 successive distns have same a at 
259 nm (ca 34.3). Accurately weigh ca 0.2 g purified nicotine; 
dissolve in and dil. to 1 L with ca 0.05/V HCI. Dil. 10 mL aliquot 
of this soln to 100 mL with ca 0.05/V HCI. Det. A at 259 nm and 
calc. a = A/{c x b), where c is concn of nicotine in g/L and b 
is cell length in cm. 



3.154 



Distillation 



Accurately weigh 2-5 g tobacco sample and transfer to distn 
flask or app. (If final detn of nicotine is gravimetric, use sample 



AOAC Methods (1980) 



Tobacco 



53 



contg ^0.1 g alkaloids; if spectrophtric, use s*2 g sample.) (If 
Griffith still is used, use 0.05-0.2 g sample.) Place 25 mL HCI 
(1+4) in receiver (1 L vol. flask is desirable) and place receiver 
so that condenser tube dips into acid. (With Griffith still, use 10 
mL HCI (1+4) in 250 mL vol. flask.) Add 50 mL alkali-salt soln 
to distn flask so that sample is rinsed into bottom of flask. (With 
Griffith still, use 5 mL alkali-salt soln.) If large vol. of fiq. is 
required for proper function of still, add more alkali-salt soln; 
do not dil. Connect flask to app. immediately and steam distil 
with as rapid current of steam as can be condensed efficiently. 
Effluent condensate should not be above room temp. Apply 
heat to distn flask from burner, mantle, or other heat source to 
keep vol. in flask approx. const. Collect ca 900 mL condensate 
(or distil addnl 100 mL after condensate shows no nicotine by 
silicotungstic acid test). (With Griffith still, collect 225 mL.) Dil. 
distillate to vol. 



3.155 



Determination 



(a) Spectrophotometry.— D\\. aliquots of distillate (if neces- 
sary) with 0.05/V HCI so that A at 259 nm is 0.5-0.8 and read A 
at 236, 259, and 282 nm. Calc. corrected^ ' 25 9 = 1 .059 x [observed 
^259 - V* t^236 +^282)] after correcting all observed A values to 
original distillate vol. basis. Concn, c, of alkaloids as nicotine in 
g/L is given bye = A' Z5g /{a x b), where a is absorptivity at 259 
nm, and b is cell length in cm. 

% alkaloid (as nicotine) = c x vol. distillate (L) x 100/g 

sample. 

(b) Gravimetric— Det. alkaloids in distillate as in 6.176, but 
double amt of silicotungstic acid specified, i.e., 2 mL/each 10 
mg alkaloids expected. 

Cundiff-Markunas Method [45)— Official Final Action 

(Total alkaloids (as nicotine), tertiary alkaloids (as nicotine), and 
secondary alkaloids (as nornicotine)) 

3.156 Reagents 

(a) Benzene-chloroform soln. — Mix equal parts by vol. of 
benzene and CHCI 3 and sat. with H 2 0. 

(b) Sodium hydroxide soln.— 36%. Dissolve 500 g NaOH in 
H 2 and dil. to 1 L. 

(c) Dilute acetic acid.—S%. Dil. 50 mL HOAc to 1 L with H 2 0. 

(d) Crystal violet indicator. — Dissolve 0.5 g crystal violet in 
100 mL HOAc. 

(e) Perchloric acid std soln.— 0.025/V. Add 4.7 mL 72% HCI0 4 
to freshly opened 5 lb bottle HOAc and mix. {Caution: See 51.022 
and 51.028(a) and (d).) Stdze as follows: Accurately weigh 0.1 g 
KH phthalate (NBS) into 125 mL erlenmeyer, add 50 mL HOAc, 
and heat to dissolve. Cool, add 2 drops indicator, and titr. to 
blue-green end point. Perform blank titrn on 50 mL HOAc and 
2 drops indicator soln, and correct vol. of titrant. 

N = wt KH phthalate x 4.896/mL HCI0 4 soln. 



3.157 



Determination 



Accurately weigh 2.5 g finely ground tobacco into 250 mL 
erlenmeyer. Add 15 mL 5% HOAc and swirl until tobacco is 
thoroly wetted. Pipet 100 mL benzene-CHCI 3 soln into flask, and 
then 10 mL 36% NaOH soln. Stopper flask tightly and shake 20 
min, using wrist-action shaker. Add 4.5-5 g (2 teaspoonfuls) 
Filter-Cel, mix, and filter most of benzene layer thru Whatman 
No. 2 paper into second flask. If filtrate has any turbidity, add 
2-2.5 g (1 teaspoonful) addnl Filter-Cel and refilter thru Whatman 
No. 2 paper. Filtrate must be clear. 

Pipet 25 mL aliquots of filtrate into each of two 125 mL 
erlenmeyers. Pass stream of air over surface of soln in first flask 



5 min, add 2 drops indicator, and titr. to green end point with 
0.025/V HCIO4. Add 1.0 mL Ac 2 to second flask and let stand 
s*15 min. Add 25 mL HOAc and 2 drops indicator, and titr. to 
blue-green end point with 0.025/V HCI0 4 . Take first appearance 
of blue-green thruout soln as end point. For each series of 
analyses perform blank titrns and correct respective vols of 
titrant. 

Calc. % alkaloids as follows: % total alkaloids (as nicotine) = 
l/,x/Vx 32.45/wt sample; % tertiary alkaloids (as nicotine) - 
{2V 2 - VJ x N x 32.45/wt sample; % secondary alkaloids (as 
nornicotine) = 2(V, - V 2 ) x N x 29.64/wt sample; where V, = 
vol. titrant for nonacetylated aliquot; V 2 - vol. titrant for acet- 
ylated aliquot; and N = normality HCI0 4 . 

Nicotine on Cambridge Filter Pads 

Gas-Liquid Chromatographic Method [46) 

Official First Action 

3.158 Apparatus and Reagents 

(a) Gas chromatograph. — With flame ionization detector, 
heated injection port, and thermostated column oven. Following 
conditions have been found satisfactory: Column, 1.8 m (6') x 
Vb" stainless steel; packing, 2% KOH and 10% Carbowax 20M 
(based on final packing wt) on 45-60 mesh calcined diat. earth 
(such as Chromosorb W, or equiv.), resieved before use to mesh 
range to remove fines and lumps; temps (°): column 165, 
detector and injection port 200-250; carrier gas flow, ca 40 
mL/min. Adjust H and air flows for max. sensitivity and stability. 
Under these conditions, column should have ht equiv. to the- 
oretical plate (HETP) <1 mm and resolution of >2, calcd with 
nicotine and anethole. 

(b) Measuring system. — Measure peak areas with electronic 
integrator or other system with resolution of ^1 count/mv-sec. 

(c) Mechanical shaker. — Capable of extg ^99% nicotine. Bur- 
rell Wrist-Action shaker has been found satisfactory. 

(d) Extracting sofn.— 2-Propanol contg 1 mg anethole/mL as 
internal std for nicotine. If H 2 is also to be detd, add 20 mg 
EtOH/mL 2-propanol as addnl internal std. 

(e) Nicotine std solns. — (/) Stock soln. — Weigh 2.500 g nicotine, 
3.153, or equiv. amt of nicotine salt. Transfer quant, into 100 mL 
vol. flask, and dil. to vol. with extg soln. (2) Working std solns. — 
Pipet 1, 2, 3, 4, and 5 mL stock soln into five 100 mL vol. flasks, 
and dil. to vol. with extg soln (0.25, 0.50, 0.75, 1.00, and 1.25 mg 
nicotine/ mL). {Caution; See precaution in 3.153.) 



3.159 



Extraction 



Place Cambridge filter material in flask or serum bottle ac- 
comodated by shaker used, add 10.00 mL extg soln, stopper, 
and shake until s*99% of nicotine is extd (usually ca 15 min). 



3.160 



Standardization 



Prime column with aliquots of 1.25 mg/mL std soln. Let 
baseline stabilize, inject 1 fit each std soln in succession, and 
repeat sequence 3 times. Det. area ratio (nicotine:anethole) for 
each injection, and calc. slope and intercept of response curve, 
preferably by method of least squares {See Definition of Terms 
and Explanatory Notes No. {24)). Correlation coefficient should 
be ^0.99 and intercept =£0.05 mg/mL 

3.161 Determination 

Prime column with aliquots of ext, 3.159. Let baseline stabilize, 
and inject 1 juL of each sample soln. Calc. nicotine concn in soln 
(C, mg/mL) = mx + b, where m = slope of stdzn curve, b = 
intercept, and x = area ratio of nicotine to anethole. 

Nicotine yield/cigaret = (Cx 10.00)/(No. cigarets/pad) 



54 



3. Plants 



AOAC Methods (1980) 



Menthol (47)— Official Final Action 
Colohmetric Method 



3.162 



Apparatus and Reagents 



(a) Distillation apparatus. — See Fig. 3:07. 
<b) Spectrophotometer. — With matched cells; capable of 
measuring A at 550 nm. 

(c) Menthol std soln. — 1 mg/mL. Accurately weigh 100 mg 
USP /-menthol into 100 mL vol. flask, add alcohol to dissolve, 
and dil. to vol. with alcohol. 

(d) DMAS color reagent. — Dissolve 0.5 g p-dimethylamino- 
benzaldehyde (Eastman Kodak, white label) in 100 mL H 2 S0 4 
(1.6+1). 

3.163 Preparation of Calibration Curve 

Prep. dil. stds by pipeting aliquots contg 0, 3, 4, 6, 8, and 10 
mg menthol into 100 mL vol. flasks and dilg to vol. with alcohol 
(1+1). Pipet 1 mL each dil. std into 10 mL test tube, add 5 mL 
color reagent, mix, and place in boiling H 2 bath exactly 2 min. 
Cool in tap H 2 0, and within 15 min det. A at 550 nm against 
std. Prep, calibration curve by plotting /4 against menthol concn 
(mg/100 mL). 



3.164 



Determination 



Accurately weigh 2.00-2.15 g cigaret filler and transfer to 
distn flask, A. Add 80 mL H 2 and few boiling stones, connect 
flask to condenser with tube, B, attach adapter, C, to condenser, 
and immerse tip in 20 mL alcohol in 100 mL vol. receiving flask. 

Gently heat distn flask until distn begins; then increase heat 
and lower receiving flask, D, so tip of adapter is no longer 
immersed. Distil until 20 mL distillate collects. Disconnect con- 
denser from tube, and wash down condenser with alcohol. 
Remove receiving flask, dil. distillate to ca 70 mL with alcohol, 
and add H 2 almost to vol. Mix, add alcohol to vol., and mix 
again. 

Pipet 1 mL distillate into 10 mL test tube, add 5 mL color 
reagent, mix, and place in boiling H 2 bath exactly 2 min. Cool 
in tap H 2 0, and within 15 min det. A at 550 nm, using "color" 
soln from nonmentholated tobacco carried thru detn as blank. 
(If nonmentholated sample corresponding to mentholated sam- 
ple is not available, use reagent blank.) Use nonmentholated 



20 MM O.D. TUBING 

f 24/40 
500 ML 




I00 ML 



FIG. 3:07— Distillation apparatus; see 3.164 for explanation of symbols 



tobacco blank within 1 5 min after color development step. Fresh 
nonmentholated tobacco blank soln may be required during 
multiple sample runs. Det. mg menthol from calibration curve. 
% Menthol = mg menthol/(g original sample x 10). 



Gas Chromatographic Method 



3.165 



Apparatus and Reagents 



(a) Gas chromatograph. — Equipped with flame ionization de- 
tector and thermostated injection port and column oven. Use 
following conditions for analysis; Column, 1.5 m (5') x %" od 
stainless steel packed with 10% (w/w) silicone oil DC-550 on 
60-80 mesh Chromosorb W; temps (°): column 150, detector 
150, injection port 175; N carrier gas flow ca 35 mL/min. Adjust 
H and air flows for max. sensitivity and reasonable stability. 

(b) Mechanical shaker. — Wrist action. 

(c) Menthol-anethole std soln. — 0.250 mg menthol and 0.50 
mg anethole/mL. Weigh exactly 0.5000 g tech. grade anethole 
and wash into 1 L vol. flask with 200 mL alcohol. Transfer 0.2500 
g USP /-menthol to the vol. flask with enough alcohol to bring 
to vol. Store soln in dark g-s bottle. Do not use >6 weeks. 

(d) Extracting so//?.— 0.50 mg anethole/mL. Dissolve 1.000 g 
anethole in alcohol in 2 L vol. flask, dil. to vol. with alcohol, and 
store in dark. 



3.166 



Determination of Ratio Factor 



Weigh ca 3 g nonmentholated control filler, contg all usual 
humectants but no menthol or anethole, into 125 mL rubber- 
stoppered flask. Pipet 50 mL std menthol-anethole soln into 
flask, stopper, and shake 1 hr on mech. shaker. Let solids settle 
15 min and chromatograph 2 /xL aliquot of supernate. Repeat 
twice more to obtain total of 3 replicates of std chromatogram. 
For quant, results, inject both std and unknown samples by 
inserting 2" (5 cm) needle to hilt, injecting 2 fxL rapidly, and 
withdrawing needle at once. (Menthol elutes in ca 3 min, 
anethole in ca 5 min.) After ca 10 min, all other compds are 
eluted and new injection can be made. 

Draw baselines under menthol and anethole peaks and meas- 
ure peak hts in mm. Using mean peak ht of menthol and 
anethole from 3 std chromatograms, calc. std ratio factor of 
menthol to anethole as follows: 

Std ratio factor - peak ht for menthol (0.25 mg/mL)/peak ht 
for anethole (0.50 mg/mL). 



3.167 



Determination 



Accurately weigh 8-8.5 g mentholated cigarette filler and 
place in 250 mL rubber-stoppered erlenmeyer. Pipet 100 mL 
extg soln into flask, stopper, and mech. shake 2 hr. Let solids 
settle 15 min and chromatograph 2 /jlL aliquot of supernate. 
Draw baselines under menthol and anethole peaks and measure 
peak hts in mm. Calc. ratio factor of unknown menthol as 
follows: 

Ratio factor for unknown = peak ht for unknown menthol/ peak 
ht for anethole (0.50 mg/mL). 

% Menthol = (unknown ratio factor x 0.25 x 10)/(std ratio 
factor x g sample). 

SELECTED REFERENCES 

(7) Botan. Gaz. 73, 44(1922); Proc. Am. Soc. Hort. Sci. 1927, p. 

191; JAOAC 13, 224(1930); 16, 71(1933); 19, 70(1936). 
(2) JAOAC 58, 436(1975). 
{3} JAOAC 11, 203(1928); 16, 70(1933); 19, 70(1936). 

(4) Ind. Eng. Chem., Anal. Ed. 9, 67(1937); 10, 13(1938); JAOAC 
25, 555(1942); 27, 526(1944). 

(5) JAOAC 19, 359(1936); 27, 526(1944). 



AOAC Methods (1980) 



Selected References 



55 



(6) J. Biol. Chem. 7,83(1910); JAOAC4,392(1921); 16,70(1933). 

(7) J. Biol. Chem. 47, 475(1921); 50, 527, 537(1922); JAOAC 
14, 216(1931); 16, 71(1933); 19, 71(1936). 

(8) JAOAC 3, 329(1920); 4, 393(1921); 16, 71(1933); 19, 71 
(1936). 

(3) JAOAC 4, 393(1921). 

(10) JAOAC 39, 419(1956). 

(11) JAOAC 19,71(1936). 

{12) J. Am. Chem. Soc. 51, 1664(1929); JAOAC 19, 71(1936). 

(13) JAOAC 34, 710(1951); 36, 405(1953). 

{14) JAOAC 36, 405(1953). 

(75) Ind. Eng. Chem., Anal. Ed. 13, 145(1941); JAOAC 24, 

520(1941). 
{16) JAOAC 36, 397(1953). 

{17) JAOAC 36, 412(1956); 41, 309(1958); 43, 511(1960). 
{18) USDA Bur. Chem. Bull. 105, p. 151; 116, p. 92; 137, p. 30. 
{19) JAOAC 6, 415(1923). 

(20) JAOAC 16,71(1933). 

(21) JAOAC 14, 216(1931); J. Biol. Chem. 59, 255(1924). 

(22) JAOAC 49, 284(1966). 

(23) JAOAC 11, 209(1928); 12, 195(1929); 21, 107(1938). 

(24) Sutton, "Systematic Handbook of Volumetric Analysis/' 
11th ed., 1924, p. 146; J. Am. Chem. Soc. 37, 1128(1915). 

(25) JAOAC 18, 379(1935); 19, 72(1936). 



(26) JAOAC 58,1 129(1975). 

(27) JAOAC 55, 991(1972); 61, 150(1978). 

(28) JAOAC 19, 236(1936). 

(29) JAOAC 52, 627(1969). 

(30) JAOAC 41, 304(1958). 

(31) JAOAC 14, 73, 225(1931); 15, 71(1932). 

(32) JAOAC 36, 402(1953). 

(33) JAOAC 41, 307, 681(1958); 42, 650(1959); 43, 512(1960); 
44,267(1961). 

(34) Anal. Chem. 20, 850(1948); JAOAC 39, 423(1956). 

(35) JAOAC 15, 124(1932); 18, 386(1935); 19, 107 (1936). 

(36) JAOAC 32, 288(1949). 

(37) Ind. Eng. Chem., Anal. Ed. 12, 148(1940); 15, 524(1943). 

(38) Ind. Eng. Chem., Anal. Ed. 14, 877(1942); JAOAC 27, 
517(1944). 

(39) Plant Physiol. 17, 198(1942). 

(40) JAOAC 49, 525(1966). 

(41) JAOAC 46, 415(1963). 

(42) JAOAC 42, 302(1959). 

(43) JAOAC 49, 521(1966). 

(44) JAOAC 54, 560(1971). 

(45) JAOAC 43, 524(1960). 

(46) JAOAC 62, 229(1979). 

(47) JAOAC 51, 650(1968). 



4. Disinfectants 



Phenol Coefficient (7) — Official Final Action 

(Applicable to testing disinfectants miscible with H 2 that do 

not exert bacteriostatic effects that cannot be neutzd by one of 

subculture media specified, or overcome by suitable subtransfer 

procedures. The 95% confidence limits are ±12%.) 



1. Using Salmonella typhi 



4.001 



Culture Media 



(a) Nutrient broth. — Boil 5 g beef ext (Difco), 5 g NaCI, and 10 
g peptone (Anatone, peptic hydrolysate of pork tissues, manu- 
factured by American Laboratories, Inc., 4410 S 102nd St, 
Omaha, NB 68127) in 1 L H 2 20 min, and dil. to vol. with H 2 0; 
adjust to pH 6.8. (If colori metric method is used, adjust broth to 
give dark green with bromothymol blue.) Filter thru paper, place 
10 mL portions in 20 x 150 mm test tubes, and autoclave 20 
min at 121°. Use this broth for daily transfers of test cultures. 

(b) Synthetic broth.—Soln A: Dissolve 0.05 g L-cystine, 0.37 
g DL-methionine, 0.4 g L-arginine.HCI, 0.3 g DL-histidine.HCI, 
0.85 g L-lysine.HCI, 0.21 g L-tyrosine, 0.5 g DL-threonine, 1 .0 g dl- 
valine, 0.8 g L-leucine, 0.44 g DL-isoleucine, 0.06 g glycine, 0.61 g 
DL-serine, 0.43 g DL-alanine, 1.3 g L-glutamic acid.HCI, 0.45 g L- 
aspartic acid, 0.26 g DL-phenylalanine, 0.05 g DL-tryptophan, and 
0.05 g L-proline in 500 mL H 2 contg 18 mL 1/V NaOH. 

So/n B: Dissolve 3.0 g NaCI, 0.2 g KCI, 0.1 g MgS0 4 .7H 2 0, 1.5 
g KH 2 P0 4 , 4.0 g Na 2 HP0 4 , 0.01 g thiamine.HCI, and 0.01 g 
niacinamide in 500 mL H 2 0. 

Mix Soins A and B, dispense in 10 mL portions in 20 x 150 
mm tubes, and autoclave 20 min at 121°. Before using for daily 
transfers of test cultures, aseptically add 0.1 mL sterile 10% 
glucose soln per tube. Grow cultures with tube slanted 8° from 
horizontal. 

(c) Nutrient agar. — Dissolve 1.5% Bacto agar (Difco) in nu- 
trient broth and adjust to pH 7.2-7 .4 (blue-green with bromo- 
thymol blue) or in synthetic broth, tube, autoclave, and slant. 

(d) Subculture media.— Use (7), (2), or (3), whichever gives 
lowest result (Com. dehydrated brands made to conform with 
preceding specifications may be used.) With oxidizing products 
and products formulated with toxic compds contg certain heavy 
metals like Hg, (2) will usually give lowest result. With products 
contg cationic surface active materials, {3) will usually give 
lowest result. See also 4.009, par. 5. 

(7) Nutrient broth described in (a); 

(2) Fluid thioglycolate medium USP XX: Mix 0.5 g L-cystine, 
0.75 g agar, 2.5 g NaCI, 5.5 g glucose.H 2 0, 5.0 g H 2 0-sol. yeast 
ext, and 15.0 g pancreatic digest of casein with 1 L H 2 0. Heat on 
H 2 bath to dissolve, add 0.5 g Na thioglycolate or 0.3 g 
thioglycolic acid, and adjust with IN NaOH to pH 7.1 ±0.2. If 
filtration is necessary, reheat without boiling and filter hot thru 
moistened filter paper. Add 1.0 mL freshly prepd 0.1% Na 
resazurin soln, transfer 10 mL portions to 20 x 150 mm tubes, 
and autoclave 20 min at 121°. Cool at once to 25° and store at 
20-30°, protected from light. 

{3) "Letheen broth": Dissolve 0.7 g lecithin (Azolectin, As- 
sociated Concentrates, 32-30 61st St, Woodside, NY 11377) and 
5.0 g polysorbate 80 (Tween 80, or equiv.) in 400 
mL hot H 2 and boil until clear. Add 600 mL soln of 5.0 g beef 
ext (Difco), 10.0 g peptone (Anatone, (a)), and 5 g NaCI in H 2 0, 
and boil 10 min. Adjust with 1/V NaOH and/or 1/V HCI to pH 7.0 
±0.2 and filter thru coarse paper; transfer 10 mL portions to 
20 x 150 mm tubes, and autoclave 20 min at 121°. 



(4) Cystine trypticase agar {BBL): Suspend 29.5 g in 1 L H 2 0. 
Heat gently with frequent agitation and boil ca 1 min or until 
soln is complete. Transfer 10 mL portions to 20 x 150 mm tubes, 
and autoclave 15 min at 12 lb pressure. Cool in upright position 
and store =£25 days at 20-30°. Use for monthly transfer of stab 
stock cultures of Ps. aeruginosa PRD 10 (ATCC 15442). 

(5) Other subculture media: Use 4.001(d)(2) with 0.7 g lecithin 
(Azolectin, Associated Concentrates, Inc., 32-30 61st St, Wood- 
side, NY 11377) and 5.0 g polysorbate 80 (Tween 80, or equiv.) 
added; or suspend 29.8 g prepd fluid thioglycolate medium 
(Difco), 0.7 g lecithin, and 5.0 g polysorbate 80 in 1 L H 2 0, and 
boil until soln is clear. Cool, dispense in 10 mL portions in 
20 x 150 mm tubes, and autoclave 20 min at 121°. Store at 
20-30°. Protect from light. 

4.002 Apparatus and Reagents 

(a) Glassware. — 1, 5, and 10 mL vol. pi pets; 1, 5, and 10 mL 
Mohr pipets graduated to 0.1 mL or less; 100 mL g-s cylinders 
graduated in 1 mL divisions; Pyrex lipped test tubes, 25 x 150 
mm (medication tubes); bacteriological culture tubes, 20 x 150 
mm (test culture and subculture tubes). Plug medication tubes 
with cotton wrapped in 1 layer of cheese cloth. Sterilize all 
glassware 2 hr in hot air oven at 180°. Loosely plug pipets with 
cotton at mouth and place in closed metal containers before 
sterilizing. 

(b) Water bath. — Insulated, relatively deep H 2 bath, with 
cover having 3=10 well-spaced holes which admit medication 
tubes but not their lips. 

(c) Racks. — Any convenient style. Blocks of wood (size de- 
pending on space in incubator) with deep holes are satisfactory. 
Have holes well spaced to ensure quick manipulation of tubes. 
It is convenient to have them large enough to admit medication 
tubes while dilns are being made. 

(d) Transfer loop. — Make 4 mm id single loop at end of 50-75 
mm (2-3") Pt or Pt alloy wire No. 23 B&S gage or 4 mm loop 
fused on 75 mm (3") shaft (available from Matthey-Bishop, Inc., 
Malvern, PA 19355). Fit other end in suitable holder (glass or Al 
rod). Bend loop at 30° angle with stem, Fig. 4:01. 

(e) Test organism. — Hopkins strain 26 of Salmonella typhi 
(Schroeter) Warren and Scott, ATCC No. 6539 (formerly called 
Bac. typhosus and Eberthella typhosa). Maintain stock culture 
on nutrient agar slants by monthly transfers. Incubate new stock 
transfer 2 days at 37°; then store at 2-5°. From stock culture 
inoculate tube of nutrient broth and make at least 4 consecutive 
daily transfers (=£30) in nutrient broth, incubating at 37°, before 
using culture for testing. (If only 1 daily transfer has been 
missed, it is not necessary to repeat the 4 consecutive transfers.) 
Use 22-26 hr culture of organism grown in nutrient broth at 37° 
in test. Shake, and let settle 15 min before using. 

With Ps. aeruginosa PRD 10, proceed as in 4.011. 

(f) Phenol stock soln.— 5% (w/v). Weigh 50 g USP phenol, 
which congeals at ^40°, in beaker. Dissolve in H 2 0, rinse soln 
into 1 L vol. flask, and dil. to vol. Stdze with 0.1/V KBr-KBr0 3 
soln, (g), as follows: Transfer 25 mL stock soln to 500 mL vol. 
flask and dil. to vol. with H 2 0. Transfer 15 mL aliquot of dild 
soln to 500 mL I flask and add 30 mL std KBr-KBr0 3 soln. Add 
5 mL HCI and immediately insert stopper. Shake frequently 
during 30 min and let stand 1 5 min. Remove stopper just enough 
to quickly add 5 mL 20% Kl soln, taking care that no Br vapors 
escape, and immediately stopper flask. Shake thoroly, remove 



56 



AOAC Methods (1980) 



Phenol Coefficient 



57 




FIG. 4:01— Transfer loop and manner of using In phenol coefficient 
technic 



stopper, and rinse it and neck of flask with little H 2 so that 
washings flow into flask. Titr. with 0.1 N Na 2 S 2 3( using starch 
indicator, 6.005(f). 1 mL 0.1/V KBr-KBr0 3 - 0.001569 g phenol. 

% phenol in stock soln = (30 - mL 0.1/V Na 2 S 2 3 soln from 

titrn) x 0.001569 x 1333 x 

100/1000; 

where 30 = mL 0.1/V KBr-KBr0 3 soln added, 0.001569 = g 

phenol equiv. to 1 mL 0.1/V KBr-KBr0 3 soln, 1333 = diln factor, 

and 1000 = original vol. phenol stock soln. 

If necessary, adjust stock soln to 5.00±0.05% phenol by 
adding H 2 or phenol. Keep in well stoppered amber bottles in 
cool place, protected from light. 

(g) Potassium bromide-bromate soln. — 0.1/V. Prep, as in 
50.020. Stdze as follows: Transfer 30 mL to I flask, and add 25 
mL H 2 0, 5 mL 20% Kl soln, and 5 mL HCI. Shake thoroly and titr. 
with 0.1/V Na 2 S 2 3 , using starch indicator, 6.005(f). 

4.003 Operating Technic 

Make 1% stock diln of substance to be tested (or any other 
convenient diln, depending on anticipated concn) in g-s cylinder. 
Make final dilns, from 1% stock diln, directly into medication 
tubes and remove all excess >5 mL. (Range of dilns should 
cover killing limits of disinfectant in 5-15 min and should at 
same time be close enough for accuracy.) From 5% stock phenol 
soln (1-20) dil. further to make 1-90 and 1-100 dilns, and place 
in medication tubes. Place these tubes, contg 5 mL each of final 
dilns of disinfectant and of phenol, and tube contg test culture 
in H 2 bath at 20° and leave 5 min. Add 0.5 mL test culture to 
each of dilns at time intervals corresponding to intervals at 
which transfers are to be made. (Thus, by time 10 tubes have 
been seeded at 30 sec intervals, 4.5 min has elapsed, and 30 sec 
interval intervenes before transference to subculture begins.) 
Add culture from graduated pipet large enough to seed alt tubes 
in any one set. In using Ps. aeruginosa PRD 10 (ATCC 15442), 
proceed as in 4.011. 

In inoculating medication tubes, hold them in slanting position 
after removal from bath, insert pipet to just above surface of 
disinfectant, and run in culture without letting tip touch disin- 



fectant. After adding culture, agitate tubes gently but thoroly to 
insure even distribution of bacteria, and replace in bath; 5 min 
after seeding first medication tube, transfer 1 loopful of mixt of 
culture and dild disinfectant from medication tube to corre- 
sponding subculture tube. To facilitate transfer of uniform drops 
of medication mixt, hold tube at 60° angle, and withdraw loop 
so that plane of loop is parallel with surface of liq. (Fig. 4:01). 
After 30 sec, transfer loopful from second medication tube to 
second subculture tube and continue process for each successive 
diln; 5 min after making first transfer, begin second set of 
transfers for 10 min period, and finally repeat for 15 min period. 
Gently agitate medication tubes before taking each interval 
loop subsample for transfer to subculture medium. Before each 
transfer, heat loop to redness in flame and flame mouth of every 
tube. Sterilize loop immediately after each transfer (before 
replugging tubes) to allow time for cooling. Use care in trans- 
ferring and seeding to prevent pipet or needle from touching 
sides or mouth of medication tube, and see that no cotton 
threads adhere to inner sides or mouths of tubes. Incubate 
subculture 48 hr at 37° and read results. Thoroly agitate individual 
subculture tubes before incubation. Macroscopic examination 
is usually sufficient. Occasionally 3-day incubation period, agar 
streak, microscopic examination, or agglutination with antity- 
phoid serum may be necessary to det. feeble growth or sus- 
pected contamination. 



4.004 



Caicuiation 



Express results in terms of phenol coefficient number, or 
highest diln killing test organism in 10 min but not in 5 min, 
whichever most accurately reflects germicidal value of disinfec- 
tant. Phenol coefficient is number obtained by dividing numerical 
value of greatest diln (denominator of fraction expressing diln) 
of disinfectant capable of killing S. typhi in 10 min but not in 5 
min by greatest diln of phenol showing same results. 

Example: 





Disinfectant 


(X): 




Diln 


5 Min 


10 Min 


15 Min 


1-300 













1-325 


+ 










1-350 


+ 










1-375 


+ 




+ 





1-400 


+ 


Phenol: 


+ 


+ 


1- 90 


+ 










1-100 


+ 




+ 


+ 



Phenol coefficient would be 



350 
90 



=3.89. 



Test is satisfactory only when phenol control gives one of 
following readings: 



Phenol 


5 Min 


10 Min 


15 Min 


1- 90 


+ or 


+ or 





1-100 


+ 


+ 


+ or 



If none of dilns of disinfectant shows growth in 5 min and 
killing in 10 min, est. hypothetical diln only when any 3 consec- 
utive dilns show following results: first, no growth in 5 min; 
second, growth in 5 and 10 min but not in 15 min; and third, 
growth in 5, 10, and 15 min. 

Example: 

Disinfectant (X): 
Diln 5 Min 10 Min 15 Min 

1-300 

1-350 + + 

1-400 + + + 

Phenol: 
1-90 

1-100 + + 

325 
Phenol coefficient would be — - =3.42. 
95 



58 



4. Disinfectants 



AOAC Methods (1980) 



To avoid giving impression of fictitious accuracy, calc. phenol 
coefficient to nearest 0.1. Thus, in examples cited above, phenol 
coefficients would be reported as 3.9 and 3.4, instead of 3.89 
and 3.42. 

Note: Although it is commonly accepted criterion that disin- 
fectants be at diln equiv. in germicidal efficiency to phenol 
against S. typhi by calcg 20 x S. typhi coefficient to det number 
of parts H 2 in which 1 part disinfectant may be mixed, this 
should be regarded as presumptive and is subject to confir- 
mation by Use-Diln Method. 

4.005 2. Using Staphylococcus aureus (?) 

Proceed as in 4.001-4.004, except change phenol dilns and 
test organisms. Use 22-26 hr culture of Staph, aureus FDA 209, 
ATCC No. 6538, having at 20° at least resistance indicated by 
following: 



Phenol 


5 Min 


10 Min 


15 Min 


1-60 


+ or 


+ or 





1-70 


+ 


+ 


+ 



Note: Calc. results as in 4.004. If conversion 20 x Staph, 
aureus coefficient is used to det. number of parts H 2 in which 
1 part germicide may be incorporated to disinfect where py- 
ogenic organisms are objective, this diln is subject to confir- 
mation by Use-Diln Method. 

4.006 3. Using Pseudomonas aeruginosa 
Official First A ction 

Proceed as in 4.001-4.004. Use 22-26 hr culture of Ps. aeru- 
ginosa PRD 10 (ATCC 15442), having resistance to phenol at 20° 
at least as follows: 



Phenol 


5 Min 


10 Min 


15 Min 


1-S0 


+ or 


+ or 





1-90 


+ 


+ 


+ 



Use-Dilution Method (2)— Official Final Action 

(Applicable to testing disinfectants miscible with H 2 to confirm 

phenol coefficient results and to det. max. dilns effective for 

practical disinfection) 



1. Using Salmonella choleraesuis 



4.007 



Reagents 



(a) Culture media. —See 4.001. 

(b) Test organism, Salmonella choleraesuis. — (ATCC 10708). 
Maintain stock culture on nutrient agar slants by monthly 
transfers. Incubate new stock transfer 2 days at 37°; then store 
at 2-5°. From stock culture inoculate tube of nutrient broth and 
incubate at 37°. Make 3 consecutive 24 hr transfers; then 
inoculate tubes of nutrient broth (2 for each 10 carriers to be 
tested), using one loop of inoculum with each tube; incubate 
48-54 hr at 37°. 

(c) Phenol.— See 4.002(f). 

(d) Sterile distilled water. — Prep, stock supply of H 2 in 1 L 
flasks, plug with cotton, sterilize 20 min at 121°, and use to prep, 
dilns of medicants. 

(e) Asparagine soin. — Make stock supply of 0.1% asparagine 
("Bacto") soln in H 2 in erlenmeyer of convenient size, plug 
with cotton, and sterilize 20 min at 121°. Use to cover metal 
carriers for sterilization and storage. 

(f) Sodium hydroxide soln. — Approx. IN (4%). (For cleaning 
metal carriers before use.) 

4.008 Apparatus 

(a) Glassware.— As in 4.002(a). Also: straight side Pyrex test 
tubes, 20 x 150 mm; 15 x 110 mm petri dishes; 100 mL, 300 



mL, and 1 L erlenmeyers. Sterilize petri dishes in closed metal 
containers. 

(b) Water bath and racks-See 4.002(b) and (c). 

(c) Transfer loops and needles.~{1) See 4.002(d). {2) Make 3 
mm right angle bend at end of 50-75 mm nichrome wire No. 18 
B&S gage. Have other end in suitable holder (glass or At rod). 

(d) Carriers. — Polished stainless steel cylinders (penicillin 
cups), 8±1 mm od, 6±1 mm id, length 10±1 mm, of type 
304 stainless steel, SS 18-8. (Obtainable from S. & L. Metal 
Products Corp., 58-29 57 Drive, Maspeth, NY 11378.) 

(e) Petri dishes. — Have available ca 6 sterile petri dishes 
matted with 2 layers of S&S No. 597 or Whatman No. 2, 9 cm 
filter paper. 

4.009 Operating Technic 

Soak ring carriers overnight in 1/V NaOH, rinse with tap H 2 
until rinse H 2 is neut. to phthln, then rinse twice with distd 
H 2 0; place cleaned ring carriers in multiples of 10 in cotton- 
plugged erlenmeyers or 25 x 150 mm cotton plugged Pyrex test 
tubes, cover with asparagine soln, 4.007(e), sterilize 20 min at 
121°, cool, and hold at room temp. Transfer 20 sterile ring 
carriers, using flamed nichrome wire hook, into 20 mL 48-54 hr 
nutrient broth test culture in sterile 25 x 150 mm medication 
tube. After 1 5 rnin contact period remove cylinders, using flamed 
nichrome wire hook, and place on end in vertical position in 
sterile petri dish matted with filter paper, 4.008(e). Cover and 
place in incubator at 37° and let dry ^20 min but ^60 min. Hold 
broth culture for detn of its resistance to phenol by phenol 
coefficient method, 4.003. 

From 5% stock phenol soln (1-20) make 1-90 and 1-100 dilns 
directly into medication tubes. Place tube for each diln in H 2 
bath and let come to 20°. Make stock soln of germicide to be 
tested in sterile g-s cylinder. From this soln make 10 mL dilns 
to be tested, depending upon phenol coefficient found and/or 
claimed against S, typhi at 20°, directly into each often 25 x 150 
mm medication tubes; place the 10 tubes in H 2 bath at 20° and 
let come to temp. Det. diln to be tested by multiplying phenol 
coefficient number found and/or claimed by 20 to det. number 
of parts H 2 in which 1 part germicide is to be incorporated. 
This detn is not required when disinfectant under test yields 
phenol coefficient that cannot be converted validly to presump- 
tive use-diln, or when analyst dets that use-diln range can be 
found without resort to phenol coefficient test. 

Add 0.5 mL of test culture suspension to 1-90 diln of phenol 
control; after 30 sec interval, add 0.5 mL to 1-100 diln of control, 
using sterile cotton-plugged pipets. After adding culture, agitate 
tubes gently but thoroly to distribute bacteria evenly, and replace 
in bath; 5 min after seeding first medication tube, transfer 1 
loopful of mixt. of culture and dild phenol from medication tube 
to corresponding subculture tube. After 30 sec, transfer loopful 
from second medication tube; 5 min after making first set of 
transfers begin second set of transfers for 10 min period; and 
finally repeat for 15 min period. Use technic of loop sampling, 
flaming loop and mouths of tubes, and agitating medication 
and subculture tubes as in phenol coefficient method, 4.003. 
Incubate subcultures 48 hr at 37° and read results. Resistance 
in 48-54 hr culture of S. choleraesuis should fall within range 
specified for 24 hr culture of 5. typhi in phenol coefficient 
method. 

Add 1 contaminated dried cylinder carrier at 1 min intervals 
to each of the 10 tubes of use-diln of germicide to be tested. 
Thus, by time 10 tubes have been seeded, 9 min will have 
elapsed, plus 1 min interval before transfer of first carrier in 
series to individual tube of subculture broth. This interval is 
const for each tube with prescribed exposure period of 10 min. 



AOAC Methods (1980) 



Available Chlorine Germicidal Equivalent 



59 



The 1 min interval between transfers allows adequate time for 
flaming and cooling nichrome wire hook and making transfer 
in manner so as to drain all excess medication from carrier. 
Flame lips of medication and subculture tubes in conventional 
manner. Immediately after placing carrier in medication tube, 
swirl tube 3 times before placing it back in bath. Thoroly shake 
subculture tubes, incubate 48 hr at 37°, and report results as + 
(growth) or - (no growth) values. 

Where there is reason to suspect that lack of growth at 
conclusion of incubation period may be due to bacteriostatic 
action of medicant adsorbed on carrier that has not been neutzd 
by subculture medium used, transfer each ring to new tube of 
sterile medium and reincubate for addnl 48 hr at 37°. Where 
soln under test is such that material adsorbed on ring carriers 
and transferred into subculture medium makes it unsuitable for 
growth of test organism, as may be case with coned acids and 
alkalies, products carrying antibiotics, and wax emulsions, trans- 
fer each ring to new tube of sterile medium 30 min after initial 
transfer and incubate both primary and secondary subculture 
tubes 48 hr at 37°. Results showing no growth on all 10 carriers 
will confirm phenol coefficient number found. Results showing 
growth on any of the 10 carriers indicate phenol coefficient 
number to be unsafe guide to diln for use. In latter case, repeat 
test, using lower dilns (higher conens) of germicide under study. 
Max. diln of germicide which kills test organism on 10 carriers 
in 10 min interval represents presumed max. safe use-diln for 
practical disinfection. 

4.010 2. Using Staphylococcus aureus 

Proceed as in 4.009 except change phenol dilns and test 
organism to those specified in 4.005. Use 48-54 hr culture of 
Staph, aureus FDA 209, ATCC No. 6538, having at least resistance 
specified for 24 hr culture at 20° in phenol coefficient method, 
4.005. Results showing growth on any of 10 carriers indicate 
that diln is too high for use in disinfecting where pyogenic 
bacteria must be killed. In such cases repeat test, using lower 
dilns (higher conens). Max. diln of germicide which kills both 
this test organism and S. choleraesuis on 10 carriers in 10 min 
interval represents max. presumed safe use-diln for disinfecting 
in hospitals, clinics, and other places where pyogenic bacteria 
have special significance. 

Note: While killing in 10 of 10 replicates specified provides 
reasonably reliable index in most cases, killing in 59 out of 60 
replicates is necessary for confidence level of 95%. 

4.01 1 3. Using Pseudomonas aeruginosa 
Official First Action 

Proceed as in 4.009. Use 48-54 hr nutrient broth culture Ps. 
aeruginosa PRD 10 (ATCC 15442). Carry stock culture on BBL 
CTA (cystine trypticase agar) in stab culture incubated 48 hr at 
37° and stored at 5° with transfer every 30 days. Transfer nutrient 
broth test cultures daily for 30-day intervals with incubation at 
37°. Make fresh transfer from stock culture every 30 days. Do 



not shake 48-54 hr test culture but decant liq. culture aseptically, 
leaving pellicle behind, to obtain 20 mL culture for inoculating 
20 carriers in medication tube. 

Available Chlorine Germicidal Equivalent 
Concentration (3) — Official Final Action 

(Applicable to H 2 0-miscible disinfectants for detg available CI 
germicidal equiv. conens with products offered for use as 
sanitizing rinses for previously cleaned nonporous surfaces, 
especially where speed of action and capacity are essential 
considerations) 

4.012 Reagents 

Use reagents specified in 4.001 and 4.002(e) and (f), and in 
addn: 

(a) Sterile distilled H 2 O.^See 4.007(d). 

(b) Sterile phosphate buffer soln. — pH 8.0. Add 97.5 mL soln 
contg 11.61 g anhyd. K 2 HP0 4 in 1 L H 2 to 2.5 mL soln contg 
9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave 20 min at 121° 
in cotton-plugged erlenmeyer. 

(c) NaOCi std stock soln. — -Approx. 5%. Store NaOCI stock 
soln in tightly closed bottle in refrigerator, and det. exact 
available CI concn at frequent intervals by As 2 3 titrn, 6.112. 

(d) Test organisms.— Use S. typhi ATCC No. 6539 or Staph, 
aureus ATCC No. 6538 or both. 



4013 
See 4.002. 



Apparatus 



4.014 Operating Technic 

Det. resistance of test culture to phenol as in 4.001-4.005, and 
use cultures with resistance specified. Prep., in sterile g-s 
cylinders, NaOCI solns contg 200, 100, and 50 ppm available CI 
in sterile buffer soln, 4.012(b). Transfer 10 mL of each soln to 25 
x 150 mm medication tubes, place tubes in 20° H 2 bath, and 
let come to temp. 

Starting with tube contg 200 ppm available CI, add 0.05 mL 
test culture prepd as in 4.002(e), shake, and return to H 2 bath. 
After 1 min, make transfer to tube of appropriate subculture 
medium, 4.001(d), using flamed 4 mm loop. At 1.5 min, add 
another 0.05 mL culture to the 200 ppm CI soln, shake, and 
return to bath. After addnl 1 min interval (2.5 min in test), make 
second subculture in same manner, and in 30 sec, or at 3 min 
time in test, add another 0.05 mL culture, shaking and returning 
to H 2 bath. After another 1 min interval (4 min in test), make 
another transfer to tube of subculture medium. 

Repeat operation to give total of 10 added increments. This 
requires total time of 14.5 min for each soln and addn of 0.5 mL 
total culture with subculture at std 1 min intervals after addn of 
culture aliquots. At conclusion of test shake all subculture tubes 
and incubate 48 hr at 37°. 

Repeat operation with solns contg 100 and 50 ppm available 
CI. Prep, soln of germicide to be tested at concn recommended 



Table 4:01 Example for Determination of Chlorine Germicidal Equivalent Concentration 















Subculture 


Series 












Concn, ppm 






















Germicide 


Avail. CI 


1 


2 


3 


4 


5 


6 


7 


8 


9 


10 




200 


_ 


_ 


_ 


_ 


- 


+ 


+ 


+ 


+ 


+ 


NaOCI control 


100 


- 


- 


- 


+ 


+ 


+ 


+ 


+ 


+ 


+ 




50 


- 


- 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 




25 


— 


— 


— 


- 


_ 


+ 


+ 


+ 


+ 


+ 


Unknown (X) 


20 


- 


- 


- 


- 


+ 


+ 


+ 


+ 


+ 


+ 




10 


- 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


+ 



= No growth 



+ = growth 



60 



4. Disinfectants 



AOAC Methods (1980) 



or selected for study in sterile H 2 in g-s graduate. Transfer 10 
mL to 25 x 150 mm medication tubes, place in H 2 bath, and 
let come to temp. Repeat operation with this soln. 

To be considered equiv. in disinfecting activity to 200 ppm 
available CI, unknown germicide must show absence of growth 
in as many consecutive tubes of subculture tube series as 200 
ppm available CI std. Det. activity equiv. to 100 and 50 ppm 
available CI in same manner. See example, Table 4:01. 

In this example, 25 ppm soln of germicide X could be 
considered equiv. to 200 ppm soln of available CI, and 20 ppm 
soln equiv. to 100 ppm of available CI, but 10 ppm soln of 
germicide X would not be considered equiv. in germicidal 
activity to 50 ppm of available CI. 

Draw conclusions relative to germicidal equiv. concns only 
when resistance of test culture to NaOCI control is such that s=1 
neg. increment is obtained at 50 ppm concn and 1 pos. increment 
is obtained at 200 ppm level. 



Sporicidal Test (4) — Official Final Action 

(Suitable for detg sporicidal activity of liq. and gaseous chems. 

Applicable to germicides for detg presence or absence of 

sporicidal activity against specified spore-forming bacteria in 

various situations and potential efficacy as sterilizing agent.) 

4.015 Reagents 

(a) Culture media.— {1) Soil extract nutrient broth.— Ext 1 lb 
garden soil in 1 L H 2 0, filter several times thru S&S No. 588 
paper, and dil. to vol. (pH should be ^5.2). Add 5 g beef ext 
(Difco), 5 g NaCI, and 10 g peptone (Anatone, 4.001(a)). Boil 20 
min, dil. to vol., adjust with 1/V NaOH to pH 6.9, and filter thru 
paper. Dispense in 10 mL portions into 25 x 150 mm tubes, and 
autoclave 20 min at 121°. Use this broth to propagate test culture 
of Bacilli. 

{2) Nutrient agar.^See 4.001(c). Use slants of this medium 
to maintain stock culture of Bacilli. 

{3) Modified fluid thiogiycoiate medium USP XX.— Prep, as 
in 4.001(d)(2), except add 20 mL 1/V NaOH to each L before 
dispensing for sterilization. Use this medium to subculture 
spores exposed to 2.5/V HCI. For spores exposed to unknown 
germicides, use fluid thiogiycoiate medium, 4.001(d)(2). 

(4) Soil extractegg-meat medium. — Add 1.5 g Bacto Egg- 
Meat Medium dehydrated (Difco) to 25 x 150 mm tube; then 
add 15 mL garden soil ext, (7), and sterilize 20 min at 121°. Use 
this medium to propagate test cultures of Clostridia and maintain 
stock cultures of species of this genus. 

(b) Test organisms. — Use Bacillus subtilis, ATCC No. 19659, 
or Clostridium sporogenes, ATCC No. 3584, for routine evalu- 
ation. Method is also applicable for use with strains of B. 
anthracis, CI. tetani, or other spore forming species. 

(c) Dilute hydrochloric acid. — 2.5/V. Use to det. resistance of 
dried spores. Stdze and adjust to 2.5/V as in 50.012. 



4.016 



Apparatus 



(a) Glassware. — Bacteriological culture tubes, unflared, 25 x 
150 mm; 100 mL g-s cylinders graduated in 1 mL divisions; 65 
mm id funnels; supply of 1 5 x 110 mm petri dishes matted with 
2 sheets 9 cm S&S No. 597 or Whatman No. 2 filter paper. 
Sterilize all glassware and matted petri dishes 2 hr in air oven 
at 180°. 

(b) Water bath.^See 4.002(b). 

(c) Racks.— See 4.002(c). 

(d) Transfer loop, hook, and forceps.— See 4.008(c). 

(e) Tissue grinder.— Arthur H.Thomas Co., No. 3431-E20, Size 
B, or equiv. 

(f) Suture loop carrier. — From spool of size 3 surgical silk 



suture, prep, std loops by wrapping the silk around ordinary 
pencil 3 times, slipping coil so formed off end of pencil, and 
holding it firmly with thumb and index finger of left hand while 
passing another piece of suture through coil, knotting, and tying 
securely. Then shear off end of coil and knotted suture to within 
2 mm. This should provide overall length of ca 65 mm of suture 
in 2-loop coil that can be conveniently handled in ordinary 
aseptic transfer, procedure. 

Ext loops in groups of 20 by immersion in 10 mL pet ether in 
stoppered, unflared test tube, shaking frequently during 30 min 
at room temp., and hold overnight (1&-24 hr) at 2-5°. Shake, 
remove loops, drain, and dry. 

(g) Cylinder carriers. — "Penicylinders," porcelain, 8±1 mm 
od, 6±1 mm id, 10±1 mm long. (Available from Fisher Scientific 
Co., No. 7-907.) Sterilize 2 hr in 180° air oven. Wash used 
Penicylinders with Triton X-100 and rinse with H 2 4 times. 

4.017 Operating Technic 

Grow all Bacilli in soil ext nutrient broth and all Clostridia in 
soil ext-meat-egg medium. Inoculate 3 tubes, using 1 loop stock 
culture, and incubate 72 hr at 37°. Place supply of suture loops 
and cylinder carriers in sep. petri dishes matted with filter paper, 
and sterilize 20 min at 121°. Use new loops for each test. 
Penicylinders must be free from chips or cracks. Filter CI. 
sporogenes thru funnel contg 2 x 5 x 5 cm sq piece of moist 
cotton or glass wool into sterile 25 x 150 mm test tubes, using 
same funnel. In prepg B. subtilis culture, pour tube of 72 hr 
culture into tissue grinder and macerate to break up pellicle. 
Filter thru sterile funnel contg moist cotton or glass wool into 
sterile 25 x 150 mm tube, repeating operation for other 2 tubes. 
Place 10 sterile suture loops or Penicylinders into each of 3 
tubes contg 10 mL filtrate from 72 hr culture of CI. sporogenes, 
agitate, and let stand 1 0-1 5 min. Using this technic, contaminate 
35 loops or cylinders. Place contaminated suture loops and/or 
cylinders into petri dish matted with 2 layers of filter paper. 
Drain. Proceed similarly for B. subtilis. 

Place the 35 suture loops or cylinders contaminated with CI. 
sporogenes or B. subtilis in vac. desiccator contg CaCI 2 and 
draw vac. of 69 cm (27") Hg for 20 min. Dry 24 hr under vac. 
(Spores dried and held under these conditions will retain re- 
sistance ^7 days.) 

Transfer 10 mL 2.5/V HCI, 4.015(c), into sterile 25 x 150 mm 
tube. Place tube in 20° const temp. H 2 bath and let come to 
temp. Rapidly transfer 4 dried, contaminated loop or cylinder 
carriers to acid tube. Transfer remaining dried, contaminated 
suture loop or cylinder carriers to tube of thiogiycoiate subcul- 
ture medium, 4.015(a)(3), as viability control. After 2, 5, 10, and 
20 min, withdraw individual loops or cylinders from acid and 
transfer to individual tubes of subculture medium. Rotate each 
tube vigorously 20 sec and resubtransfer. Incubate 21 days at 
37 p . Test spores should resist HCI 5=2 min, and many may resist 
HCI for full 20 min. 

When testing sporicidal or sterilizing activity of gas, place 
carriers in polyethylene bags or in petri dishes with lids ajar. 
Certain gases may require rehydration of spores before exposure 
to gas. Rehydrate spores on carriers by 1 hr immersion in H 2 0, 
using ^20 mL H 2 0/6 carriers. Drain carriers 20 min on petri 
dishes matted with filter paper. After exposure to gas, remove 
carriers, using aseptic technic to subculture media as specified 
in next par. 

For aq. sporicides and sterilizers, place 10 mL product at diln 
recommended for use or under investigation into each of six 25 
x 150 mm tubes. Place tubes in 20° H 2 bath and let come to 
temp. Using flamed forceps, place 5 suture loops or cylinders, 
contaminated with Cf. sporogenes or B. subtilis and dried 24 hr 



AOAC Methods (1980) 



Sanitizers 



61 



under vac, into each of the 6 tubes contg disinfectant using 2- 
min intervals for seeding each tube. Five suture loops or 
cylinders can be placed into each tube within 5 sec. This seeding 
operation will take 10 min. After contact period specified for 
disinfectant has been achieved, remove suture loops or cylin- 
ders, using sterilized needle hook, from each tube of disinfectant 
to subculture medium or other subculture medium specified in 
4.001(d) (select medium contg most suitable neutralizer), placing 
1 suture loop or cylinder per tube. Five cylinders can be removed 
within each 2 min interval. Flame transfer needle hook after 
each carrier has been transferred to subculture medium. After 
completing transfer, resubtransfer each suture loop or cylinder 
to fresh tube of thioglycolate medium and incubate 21 days at 
37°. If no growth is observed after 21 days, heat-shock tubes 20 
min at 80° and reincubate 72 hr at 37°. Report results as + 
(growth) or - (no growth) values. 

Killing in 59 of 60 replicates on 1 carrier at diln and time 
specified is considered evidence of sporicidal efficacy against 
1 test spore and for confidence level of 95%. Tests with both B. 
subtilis and CI. sporogenes, using 30 replicates with each of 2 
carriers specified to provide min. of 120 carriers, are required 
to presumptively support unqualified sporicidal claim or for 
presumptive evidence of sterilizing activity at concn, time, and 
conditions specified. For sporicidal claims, no more than 2 
failures can be tolerated in this 120 carrier trial. For sterilizing 
claims, no failures can be tolerated. 

Fungicidal Test (5) — Official Final Action 

(Applicable for use with H ? 0-miscible type 
fungicides used to disinfect inanimate objects) 

Using Trichophyton mentagrophytes 



4.018 



Test Organism 



Use as test fungus typical strain of Trichophyton mentagro- 
phytes isolated from dermatophytes of foot. Strain must 
sporulate freely on artificial media, presence of abundant conidia 
being manifested by powdery appearance on surface of 10-day 
culture, particularly at top of agar slant, and confirmed by 
microscopic examination. Conidia-bearing mycelium should 
peel easily from surface of glucose agar. Conidia of required 
resistance survive 10 min exposure at 20° to phenol diln of 1:70, 
but not to one of 1 :60. Strain No. 640, ATCC No. 9533, is suitable. 



4.019 



Culture Medium 



Carry fungus on agar slants of following composition: Glucose 
2%, Neopeptone (Difco) 1%, agar 2%, adjusted to pH 6.1-6.3. 
Use same culture medium to prep, cultures for obtaining conidia! 
suspension, and use fluid medium of same nutrient composition 
(without agar) to test viability of conidia after exposure to 
fungicide. 



4.020 



Care of Fungus Strain 



Store stock culture of fungus on glucose agar slants at 2-5°. 
At intervals ^3 months, transfer to fresh agar slants, incubate 
10 days at 25-30°, and store at 2-5° until next transfer period. 
Do not use culture that has been kept at or above room temp. 
>10 days as source of inoculum for culture. (Cultures may be 
kept at room temp, to preserve strain and to inoculate cultures 
if transferred at intervals ^10 days.) 

4.021 Preparation of Conidia/ Suspension 

Prep, petri dish cultures by planting inoculum at center of 
agar plate and incubating culture at 25-30° for ^10, but =S15 
days. Remove mycelial mats from surface of 5 agar plate 



cultures, using sterile spatula or heavy flattened wire. Transfer 
to heat-sterilized glass tissue grinder, 4.016(e), and macerate 
with 25 mL sterile physiological NaCI soln (0.85% NaCI), or to 
heat-sterilized erlenmeyer contg 25 mL sterile saline with glass 
beads, and shake thoroly. Filter suspension thru sterile absorbent 
cotton to remove hyphal elements. Est. density of conidial 
suspension by counting in hemacytometer and store at 2-10° 
as stock spore suspension (125-155 x 10 6 conidia/mL) for ^4 
weeks for use in prepg test suspensions of conidia. Stdze test 
conidial suspensions as needed by dilg stock spore suspension 
with physiological NaCI soln so that it contains 5 x 10 6 
conidia/mL. 

4.022 Operating Technic 

Prep, dilns of fungicide. (Tests are similar to those described 
in 4.003.) Place 5 mL of each fungicide soln and of phenol 
control solns in 25 x 150 mm test-culture tubes, arrange in order 
of ascending dilns, place tubes in 20° H 2 bath, and let come to 
temp. With graduated pipet place 0.5 mL spore suspension in 
first tube of fungicidal soln, shake, and immediately replace in 
H 2 bath; 30 sec later add 0.5 mL conidial suspension to second 
tube. Repeat at 30 sec intervals for each fungicidal diln. If more 
convenient, run test at 20 sec intervals. After 5, 10, and 15 min 
exposure to fungicide, remove sample from each conidia-fun- 
gicide mixt with 4 mm loop and place in 10 mL glucose broth, 
4.019. To eliminate risk of faulty results due to possible fungi- 
static action, make subtransfers from the initial glucose broth 
subculture tubes to fresh tubes of glucose broth, using the 4 
mm loop before incubation, or make initial subcultures in 
glucose broth contg either 0.05% Na thioglycolate, 1.5% iso- 
octylphenoxy-polyethoxy-ethanol, or mixt. of 0.07% lecithin 
(Azolectin, Associated Concentrates, Inc., 32-30 61st St Wood- 
side, NY 1 1377) and 0.5% polysorbate 80 (Tween 80), whichever 
gives lowest result. Incubate inoculated tubes at 25-30°. Read 
final results after 10 days, altho indicative reading can be made 
in 4 days. 

Note: Highest diln that kills spores within 10 min is commonly 
considered as highest diln that could be expected to disinfect 
inanimate surfaces contaminated with pathogenic fungi. 

Germicidal and Detergent Sanitizers [6) 
Official Final Action 

(Suitable for detg min. concn of chem. that can be permitted for 
use in sanitizing precleaned, nonporous food contact surfaces. 
Min. recommended starting concn is 2— 4x this concn. Test also 
dets max. water hardness for claimed concns. As control, check 
accuracy of hard-water tolerance results with pure C 14 afkyl 
dimethyl benzyl NH 4 chloride (Onyx Chemical Co. 190 Warren 
St, Jersey City, NJ 07302) at 700 and 900 ppm hardness, and 
pure C 16 alkyl dimethyl benzyl NH 4 chloride (Cetalkonium Chlo- 
ride, Sterling Chemical Co.), at 400 and 550 ppm hardness, 
expressed as CaC0 3 .) 

4.023 Reagents 

(a) Culture media. — (7) Nutrient agar A. — Boil 3 g beef ext, 5 
g peptone (Bacto or equiv.; special grades must not be used), 
and 15 g salt-free agar in 1 L H 2 0. Do not use premixed, 
dehydrated media. Tube, and autoclave 20 min at 121°. Use for 
daily transfer of test culture. {2) Nutrient agar B. — Prep, as above 
but use 30 g agar. Use for growing test cultures in French square 
bottles. (3) Nutrient agar {AOAC).— See 4.001(c). Use for prepg 
stock culture slants. 

(b) Subculture media. — (7) Use tryptone glucose ext agar 
(Difco), adding 25 mL stock neutralizer, (c)/L. (2) Tryptone 
glucose ext agar (Difco). 

(c) Neutralizer stock soln. — Mix 40 g Azolectin (Associated 
Concentrates, 32-30 61st St, Woodside, NY 11377), 280 mL 



62 



4. Disinfectants 



AOAC Methods (1980) 



polysorbate 80, and 1.25 ml_ phosphate buffer, (e); dil. with H 2 
to 1 L and adjust to pH 7.2. Dispense in 100 mL portions and 
autoclave 20 min at 121°. 

(d) Neutralizer blanks.— for use with ^200 ppm quaternary 
NH 4 compd. Mix 100 mL neutralizer stock soln, (c), 25 mL 0.25/W 
phosphate buffer stock soln, (e), and 1675 mL H 2 0. Dispense 9 
mL portions into 20 x 150 mm tubes. Autoclave 20 min at 121°. 

(e) Phosphate buffer stock soln. — 0.25M. Dissolve 34.0 g 
KH 2 P0 4 in 500 mL H 2 0, adjust to pH 7.2 with 1/V NaOH, and dil. 
to 1 L. 

(f) Phosphate buffer dilution wafer.— Add 1.25 mL 0.25/W 
phosphate buffer stock soln, (e), to 1 L H 2 and dispense in 99 
mL portions. Autoclave 20 min at 121°. 

(g) Test organisms. — Use Escherichia coif ATCC No. 1 1 229 or 
Staphylococcus aureus ATCC 6538. Incubate 24 and 48 hr, res p. 
Maintain stock cultures on nutrient agar (AOAC), (a)(3), at 
refrigerator temp. 



4.024 



Res/stance to Phenol of Test Cultures 



Det. resistance to phenol at least every 3 months by 
4.001-4.005. Resistance of E. coli should be equiv. to that 
specified for S. typhi in 4.004 and that for Staph, aureus equiv. 
to that specified for this organism in 4.005. 

4.025 Apparatus 

(a) Glassware. —250 mL wide-mouth erlenmeyers; 100 mL 
graduate; Mohr, serological, and/or bacteriological (APHA spec- 
ification) pipets; 20 x 150 mm test tubes. Sterilize at 180° in hot 
air oven 3=2 hr. 

(b) Petri dishes. — Sterile. 

(c) French square bottles. — 175 mL, borosilicate. Use of other 
containers will give variable results. 

(d) Water bath.— Controlled at 25°. 



4.026 



Preparation of Culture Suspension 



From stock culture inoculate tube of nutrient agar A, 
4.023(a)(7), and make 5=3 consecutive daily transfers (=s30), 
incubating transfers 20-24 hr at 35-37°. Do not use transfers 
>30 days. If only 1 daily transfer has been missed, no special 
procedures are required; if 2 daily transfers are missed, repeat 
with 3 daily transfers. 

Prep. 175 mL French square culture bottles contg 20 mL 
nutrient agar B, 4.023(a)(2), autoclave 20 min at 121°, and let 
solidify with bottle in horizontal position. Inoculate culture 
bottles by washing growth from slant with 5 mL phosphate 
buffer diln H 2 0, 4.023(f), into 99 mL phosphate buffer diln H 2 0, 
and adding 2 mL of this suspension to each culture bottle, tilting 
back and forth to distribute suspension; then drain excess Mq. 
Incubate 18-24 hr at 35-37°, agar side down. Remove culture 
from agar surface of 4 or more bottles, using 3 mL phosphate 
buffer diln H 2 and glass beads in each bottle to suspend 
growth. Filter suspension thru Whatman No. 2 paper prewet 
with 1 mL sterile phosphate buffer, and collect in sterile tube. 



(To hasten filtration, rub paper gently with sterile policeman.) 
Stdze suspension to give av. of 10 x 10 9 organisms/mL by diln 
with sterile phosphate buffer diln H 2 0, 4.023(f). 

If Lumetron colorimeter is used, dil. suspension in sterile 
Lumetron tube to give % T according to Table 4:02. 

If McFarland nephelometer and BaS0 4 stds are used, select 
7 tubes of same id as that contg test culture suspension. Place 
10 mL of each suspension of BaS0 4 , prepd as indicated in Table 
4:03, in each tube and seal tube. Stdze suspension to correspond 
to No. 4 std. 

Table 4:03 Preparation of BaS0 4 Suspensions Corresponding 
to Bacterial Concentrations 





mL 2% BaCI 2 


mL 1% H 2 S0 4 


Av. Bacterial 


Std No. 


Soln 


(v/v) Soln 


Count/mL 


1 


4.0 


96.0 


5.0 x 10 9 


2 


5.0 


95.0 


7.5 


3 


6.0 


94.0 


8,5 


4 


7.0 


93.0 


10.0 


5 


8.0 


92.0 


12.0 


6 


10.0 


90.0 


13.5 


7 


12.0 


88.0 


15.0 



4.027 



Synthetic Hard Water 



Prep. Soln 1 by dissolving 31.74 g MgCI 2 (or equiv. of hydrates) 
and 73.99 g CaCI 2 in boiled distd H 2 and dilg to 1 L Prep. Soln 
2 by dissolving 56.03 g NaHC0 3 in boiled distd H 2 and dilg to 
1 L. Soln 1 may be heat sterilized; Soln 2 must be sterilized by 
filtration. Place required amt Soln 7 in sterile 1 L flask and add 
2=600 mL sterile distd H 2 0; then add 4 mL Soln 2 and dil. to 1 
L with sterile distd H 2 0. Each mL Soln 1 will give a water equiv. 
to ca 100 ppm of hardness calcd as CaC0 3 by formula: 

Total hardness as ppm CaC0 3 

= 2.495 x ppm Ca + 4.115 x ppm Mg. 

pH of all test waters =£2000 ppm hardness should be 7.6-8.0. 
Check prepd synthetic waters chemically for hardness at time 
of tests, using following method or other methods described in 
14th Ed. of Standard Methods for the Examination of Water, 
Sewage, and Industrial Wastes. 



4.028 



Hardness Method 



(a) EDTA std soln. —Dissolve 4.0 g Na 2 H 2 EDTA.2H 2 and 0.10 
g MgCI 2 .6H 2 in 800 mL H 2 and adjust by subsequent diln so 
that 1 mL of soln is equiv. to 1 mg CaC0 3 when titrd as in (c). 
Check EDTA soln after prepn or, if com. purchased, against 
CaC0 3 std at least every 2 months. 

(b) Calcium std soln.—*\ mL = 1 mg CaC0 3 . Weigh 1.00 g 
CaC0 3 , dried overnight or longer at 1 05°, into 500 mL erlenmeyer 
and add dil. HCI thru funnel until CaC0 3 is dissolved. Add 200 
mL H 2 0, boil to expel C0 2 , and cool. Add few drops Me red 
indicator and adjust color to intermediate orange with dil. NH 4 OH 
or HCI as required. Transfer quant, to 1 L vol. flask and dil. to 
vol. 



Table 4:02 Per Cent Light Transmission at Various Wavelengths Corresponding to Bacterial Concentrations 







% Light Transm 


ission 


with Filters, nm 






Av. Bacterial 


370 


420 


490 


530 


550 


580 


650 


Count/mL 


7.0 


4.0 


6.0 


6.0 


6.0 


7.0 


8.0 


13.0 x 10 9 


8.0 


5.0 


7.0 


7.0 


7.0 


8.0 


9.0 


11.5 


9.0 


6.0 


8.0 


8.0 


8.0 


9.0 


10.0 


10.2 


10.0 


7.0 


9.0 


9.0 


9.0 


11.0 


11.0 


8.6 


11.0 


8.0 


10.0 


10.0 


10.0 


12.0 


13.0 


7.7 


13.0 


9.0 


12.0 


12.0 


12.0 


13.0 


15.0 


6.7 



AOAC Methods (1980) 



Germicidal Sprays 



63 



(c) Determination.— Oil. 5-25 mL sample (depending on hard- 
ness) to 50 mL with H 2 in erlenmeyer or casserole. Add 1 mL 
buffer soln (67.5 g NH 4 CI and 570 mL NH 4 OH dild to 1 L with 
H 2 0), 1 mL inhibitor (5.0 g Na 2 S.9H 2 or 3.7 g Na 2 S.5H 2 
dissolved in 100 mL H 2 0), and 1 or 2 drops indicator soln (0.5 
g Chrome Black T in 100 mL 60-60% alcohol). Titr. with EDTA 
std soln slowly, stirring continuously, until last reddish tinge 
disappears from soln, adding last few drops at 3—5 sec intervals. 
Hardness as mg CaC0 3 /L = (mL std soln x 1000)/mL sample. 

4.029 Preparation of Samples 

Use composition declared or detd as guide to sample wt 
required for vol. sterile H 2 used to prep. 20,000 ppm soln. From 
this stock dtln, transfer 1 mL into 99 mL of the water to be used 
in test to give concn of 200 ppm. In making transfer, fill 1 mL 
pipet and drain back into stock soln; then refill, to correct for 
adsorption on glass. After mixing, discard 1 mL to provide 99 
mL of the test water in 4,030. 

4.030 Operating Technic 

Measure 99 mL water to be used in test, contg bactericide at 
concn to be tested, into chem. clean, sterile, 250 mL wide-mouth 
erlenmeyer and place in const temp, bath until it reaches 25°, 
or s=20 min. Prep, duplicate flasks for each germicide to be 
tested. Also prep, similar flask contg 99 mL sterile phosphate 
buffer diln H 2 0, 4.023(f), as "initial numbers" control. 

Add 1 mL culture suspension to each test flask as follows: 
Whirl flask, stopping just before suspension is added, creating 
enough residual motion of liq. to prevent pooling of suspension 
at point of contact with test water. Add suspension midway 
between center and edge of surface with tip of pipet slightly 
immersed in test soln. Avoid touching pipet to neck or side of 
flask during addn. Transfer 1 mL portions of this exposed culture 
to neutralizer blanks exactly 30 and 60 sec after addn of 
suspension. Mix well immediately after transfer. 

For "numbers control" transfer, add 1 mL culture suspension 
to 99 mL sterile phosphate diln H 2 in same manner. In case of 
numbers control, plants need be made only immediately after 
adding and mixing thoroly =s30 sec. (It is advantageous to use 
milk pipets to add culture and withdraw samples.) 

Plate from neutralizer tube to agar, using subculture medium 
4.023(b)(7) for quaternary NH 4 compds and 4.023(b)(2) with 
numbers control. Where 0.1 mL portions are planted, use 1 mL 
pipet graduated in 0.1 mL intervals. For dilns to give countable 
plates, use phosphate buffer diln H 2 0, 4.023(f). For numbers 
control, use following diln procedure: Transfer 1 mL exposed 
culture (1 mL culture suspension transferred to 99 mL phosphate 
buffer diln H 2 in H 2 bath) to 99 mL phosphate buffer diln H 2 0, 
4.023(f), {diin 1). Shake thoroly and transfer 1 mL diln 1 to 99 mL 
phosphate buffer diln H 2 0, 4.023(f), {diin 2). Shake thoroly and 
transfer 1 mL diin 2 to 99 mL phosphate buffer diln H 2 {diin 3). 
Shake thoroly and transfer four 1 mL and four 0.1 mL aliquots 
from diln 3 to individual sterile petri dishes. 

For test samples, use following diln procedure: Transfer 1 mL 
exposed culture into 9 mL neutralizer, 4.023(d). Shake and 
transfer four 1 mL and four 0.1 mL aliquots to individual sterile 
petri dishes. For numbers control, use subculture medium 
4.023(b)(2); for tests with quaternary NH 4 compds, use medium 
4.023(b)(7). Cool agar to solidify, and then invert and incubate 
48 hr at 35° before counting. 



4.031 



Results 



To be considered valid, results must meet std effectiveness: 
99.999% reduction in count of number of organisms within 30 



sec. Report results according to actual count and % reduction 
over numbers control. Counts on numbers control for germicide 
test mixt. should fall between 75 and 125 x 10 6 /mL for % 
reductions to be considered valid. 

4.032 Sterility Controls 

(a) Neutralizer. — Plate 1 mL from previously unopened tube. 

(b) Water. — Plate 1 mL from each type of water used. 

(c) Sterile distilled water. — Plate 1 mL. 

After counting plates, confirm that surviving organisms are 
E. coli by transfer to brilliant green bile broth fermentation tubes 
or lactose broth and EMB agar; confirm Staph, aureus by 
microscopic examination. 



Germicidal Spray Products (7) — Official Final Action 

(Suitable for detg effectiveness of sprays and pressurized spray 
products as spot disinfectants for contaminated surfaces) 

4.033 Reagents 

Use culture media and reagents specified in 4.001, 4.002(e) 
and (f), and 4.007 except that test organism Salmonella typhi is 
not used. 

Use as test organisms Trichophyton mentagrophytes ATCC 
No. 9533, prepd as in 4.021, to which has been added 0.02 mL 
octylphenoxy-polyethoxy-ethanol (Triton X100, Rohm & 
Haas)/10 mL suspension to facilitate spreading, Salmonella 
choleraesuis ATCC No. 10708, 4.007(b), Staphylococcus aureus 
ATCC No. 6538, maintained as in 4.007(b), and Pseudomonas 
aeruginosa ATCC No. 15442, maintained as in 4.011. 

4.034 Apparatus 

Use app. specified in 4.002 and 4.008, and in addn: 

(a) Capillary pipets. — 0.1 mL, graduated to deliver 0.01 mL. 
Sterilize in air oven 2 hr at 180°. 

(b) Microscope slides. — Non-corrosive, 25 x 25 mm (1 x 1"), 
or 18 x 36 mm glass slide. Sterilize by placing individual slides 
in petri dish matted with 2 pieces 9 cm filter paper (Whatman 
No. 2, or equiv.) in air oven 2 hr at 180°. 

(c) Bacteriological culture tubes. — Pyrex, 32 x 200 mm. 

(d) Metal forceps. — Sharp points, straight, 115 mm long. 

4.035 Operating Technic 

Thoroly shake 48 hr nutrient broth cultures of S. choleraesuis 
and Staph, aureus and let settle 10 min. With sterile capillary 
pipet or sterile 4.0 mm loop, transfer 0.01 mL culture onto 1 sq 
in. sterile test slide in petri dish and immediately spread uni- 
formly over entire area. Cover dish immediately and repeat 
operation until 12 slides have been prepd for each organism. 
(Use 2 slides as control.) Dry all slides 30-40 min at 37°. 

Spray 10 slides for specified time and distance. Hold each 
slide 10 min, drain off excess liq., and transfer slide to individual 
32 x 200 mm tube contg 20 mL appropriate subculture medium, 
4.001(d), with flamed forceps. Shake culture thoroly. If broth 
appears cloudy after 30 min, make subculture to fresh individual 
tubes of subculture broth. Transfer 2 unsprayed slides, as 
viability controls, to individual subculture tubes in same manner. 

Incubate all tubes used for primary and secondary transfers 
48 hr at 37°. Read as + (growth) or - (no growth). Killing of test 
organisms in 10 of 10 trials is presumptive evidence of disin- 
fecting action. 

Det. resistance of S. choleraesuis as in 4.003; with S. aureus 
as in 4.005; with Ps. aeruginosa as in 4.006; and with T. 
mentagrophytes as in 4.018. 



64 



4. Disinfectants 



AOAC Methods (1980) 



If there is reason to believe that lack of growth in subtransfer 
tubes is due to bacteriostasis, inoculate all incubated subculture 
tubes with loop needle inoculation of respective test culture and 
reincubate. Growth of these inocula eliminates bacteriostasis as 
cause of lack of growth. If there is question as to possibility of 
contamination as source of growth in subculture tubes, make 
gram stains and/or subculture for identification, according to 
respective test culture. 

If fungicidal activity as well as germicidal activity is involved, 
use test suspension of T. mentagrophytes spores, 4.021, and 
prep. 12 slides, using 0.01 mL std spore suspension, spraying 
and subculturing exactly as above. Make subcultures in glucose 
broth, 4.019, incubating 7 days at 25-30°. 



Water Disinfectants for Swimming Pools [8) 
Official Final Action 

(Suitable for presumptive evidence of acceptability of products 
for disinfecting swimming pool water) 



4.040 



Reagents 



4.036 



Test Culture Media 



(a) Nutrient Agar A.— See 4.023(a)(7). 

(b) Nutrient Agar B (Trypticase Soy Agar, BBL). -See 4.037(b). 

(c) Nutrient Agar C — Prep, as in 4.001(c). 



4.037 



Subculture Media 



(a) Tryptone glucose extract agar {Difco). — Dissolve 24 g in 
1 L freshly distd H 2 and heat to bp to dissolve completely. 
Autoclave 15 min at 121°. Use for plate counts of E. co// survivors. 

(b) Trypticase soy agar (BBL). — Suspend 40 g powder in 1 L 
H 2 0. Let stand 5 min and mix thoroly. Heat gently with occasional 
agitation and boil ca 1 min or until soln is complete. Autoclave 
15 min at 121°. Let cool and reautoclave 15 min at 121°. Use for 
plate counts of S. faecafis survivors. 

(c) Fluid thioglycolate medium (Difco).— See 4.001(d)(2). 

(d) Lactose broth (Difco). — Dissolve 19 g in 1 L H 2 0. Dispense 
10 mL portions into tubes with fermentation vials. Autoclave 15 
min at 121°. Use for detg presence off. coli survivors. 

(e) Eosin methylene blue agar (Difco). — Suspend 36 g in 1 L 
H 2 and heat to bp to dissolve completely. Autoclave 15 min at 
121°. Use for confirming £ coli survivors. 

(f) S-F agar (Difco).— Dissolve 36 g in 1 L H 2 0. Add 15 g agar 
and heat to bp to dissolve completely. Autoclave 15 min at 121°. 
Use for confirming S. faecalis survivors. 



4.038 



Neutralizer Stock Solns 



(a) Sodium th/osulfate soln. — Dissolve 1 g Na 2 S 2 3 in 1 L H z O. 
Dispense in 100 mL portions and autoclave 20 min at 15 lb. 

(b) Azoiectin soln.— See 4.023(c). 

(c) Other preparations. — Prepns found to be suitable and 
necessary, depending upon nature of germicidal prepns to be 
tested. 



4039 



Neutralizer Blanks 



(a) With 0.6 ppm residual chlorine or less.— Di I. 10 mL neu- 
tralizer stock soln, 4.038(a), with 90 mL sterile H 2 0. Dispense 
aseptically in 9 mL portions into sterile 25 x 150 mm tubes. 

(b) With quaternary ammonium compounds and phenolic 
derivatives. — Mix 10 mL neutralizer stock soln, 4.038(b), 2.5 mL 
0.25/W phosphate buffer stock soln, 4.040(a), and 167.5 mL H 2 0. 
Dispense in 9 mL portions into 20 x 150 mm tubes. Autoclave 
20 min at 121°. 

(c) Other preparations.— Use dilns of 4.038(c) as suitable. 



(a) Phosphate buffer stock soln.— 0.2BM. See 4.023(e). 

(b) Phosphate buffer dilution water. — See 4.023(f). 

(c) Sodium thiosuifate std solns. — (1) 0.1 N. Dissolve exactly 
24.820 g Na 2 S 2 3 .5H 2 in H 2 and dil. to 1 L. Stdze as in 50.038. 

(2) 0.001N—DW. 10 mL soln (7) to 1 L with H 2 0. 

(d) Starch indicator soln. — See 6.005(f), except use few drops 
CHCI 3 instead of Hg as preservative. 

(e) Sterile phosphate buffer stock solns. — (7) Dissolve 11.61 
g anhyd. K 2 HP0 4 in 1 L H 2 and autoclave 20 min at 121°. (2) 
Dissolve 9.08 g anhyd. KH 2 P0 4 in 1 L H 2 and autoclave 20 min 
at 121°. 

(f) NaOCI stock soln.— Approx. 5%. Store NaOCI stock soln 
in tightly closed bottle in refrigerator and det. exact available CI 
at frequent intervals by As 2 3 titrn, 6.112. 

(g) Test organism. — Use Escherichia coli ATCC 11229 and 
Streptococcus faecalis PRD {Microbiology Lab., Benefits and 
Field Services Div., EPA, Beltsville, MD 20705). Maintain, by 
monthly transfer, stock cultures of E. coli on Nutrient Agar C, 
4.001(c), and S. faecalis on Nutrient Agar B, 4.037(b); store at 
4-5°. 



4.041 



Apparatus 



(a) Glassware. — 500 mL wide- mouth erlenmeyers; 100 mL 
graduates; Mohr pipets; milk pipets; 20 x 150 mm tubes; Board 
of Health tubes; 200, 500, and 1000 mL vol. flasks. Wash in 
strong, fresh chromic acid cleaning soln, and fill and drain with 
H 2 ^3 times. Heat ^2 hr at 180° in hot air oven. 

(b) Petri dishes. — Sterile. 

(c) Water bath.— Controlled at 20 or 25°. 

4.042 Preparation of Culture Suspension 

From stock culture, inoculate tube Nutrient Agar A for E. coli 
and Nutrient Agar B for S. faecalis; make ^3 consecutive daily 
transfers «30), incubating transfer 20-24 hr at 35-37°. Do not 
transfer >30 days. If only 1 daily transfer has been missed, 
no special procedures are required; if 2 daily transfers are 
missed, repeat with 3 daily transfers. Remove culture from agar 
surface, using 5 mL phosphate buffer diln H 2 0, 4.040(b). Transfer 
culture suspension to sterile centrf. tube and centrf. 1-2 min at 
speed necessary to settle agar particles. Transfer supernate to 
another sterile centrf. tube and centrf. to obtain complete sepn 
of cells. Discard supernate and resuspend cells in 5 mL buffer 
diln H 2 0. With S. faecalis, centrf., discard supernate, and resus- 
pend cells in 5 mL buffer diln H 2 2 addnl times. Finally, stdze 
suspension to give av. of 2.0 x 10 8 organisms/mL by diln with 
sterile phosphate diln H 2 0. 

If Lumetron is used, dil. suspension in sterile Lumetron tube 
to give % T according to Table 4:04. Make serial diln plate count 
of each culture suspension before use, using phosphate buffer 
diln H 2 0, 4.040(b), and subculture medium, 4.037(a), with E. coli, 
and (b) with S. faecalis. Incubate diln plates in inverted position 
48 hr at 35-37°. Use Quebec Colony Counting Chamber and 
report results in terms of number of bacteria/ mL suspension. 

Table 4:04 Per Cent Light Transmission at Various Wave- 
lengths Corresponding to Bacterial Concentrations 







% Light Transmission 
with Filter, nm 




Av. 

Bacterial 

Count 

/mL 




370 


420 490 530 580 


650 


E. coli 
S. faecalis 


90 
86 


88 89 88 91 
82 85 85 87 


92 
89 


2.0 x 10 8 

2.0 x 10 8 



AOAC Methods (1980) 



TUBERCULOCIDAL ACTIVITY 



65 



Count of 2.0 x 10 8 is desired so that 1 mL test culture suspension 
+ 199 mL test soln will provide solncontgl x 10 6 organisms/ mL. 
Permitted variation in test culture suspension is +500,000 and 
-100,000/mL of 200 mL test soln. Use actual count for calcg 
zero time count in later tests. 

4.043 Determining Chlorine Demand of 

Freshly Distilled Test Water 

Place 200 mL H 2 in each of five 500 mL erlenmeyers. To 
flasks 1-5, resp., add 0.025, 0.05, 0.075, 0.1, and 0.15 mL of 200 
ppm available CI prepd from NaOCI soln, 4.040(f). Shake each 
flask, and let stand several min. Add crystal Kl and 1 mL HOAc, 
and swirl. Add 1 mL starch soln, 4.040(d). Flask showing 
perceptible blue indicates CI demand has been satisfied. 

4.044 Operating Technic 

Place ca 600 mL freshly sterilized distd H 2 in 1 L vol. flask. 
Add ca 1 .5-3.0 mL K 2 HP0 4 buffer, 4.040(e)( 7), and 0.5 mL KH 2 P0 4 , 
4.040(e)(2), and dil. to 900 mL Add enough NaOCI from suitable 
diluent of std stock soln, 4.040(f), to satisfy CI demand of 1 L 
test H 2 0, 4.043, and to provide ca 0.6 ppm residual available CI. 
Dil. to vol. (Example: If CI demand of H 2 is 0.1 ppm, add 3.5 
mL of 200 ppm soln of available CI made from std stock NaOCI 
soln, 4.040(f), and dil. to vol. This should provide soln with ca 
0.6 ppm residual available CI at pH 7.5 ± 0.1.) Transfer 199 mL 
of this test soln to each of three 500 mL erlenmeyers and place 
in H 2 bath at either 20 or 25°. Let come to temp. 

To first flask, add 1 mL boiled distd H 2 and det. residual 
available CI as follows: Add small crystal Kl and 1 mL HOAc; 
then add 1 mL starch soln, 4.040(d). Blue soln indicates presence 
of CI. Titr. with 0.001/V Na 2 S 2 3 , 4.040(c)(2), until color disap- 
pears; mL 0.001/V Na 2 S 2 3 x 0.1773 = ppm residual available 
CI. This represents available CI at time in test. Result should 
be 2*0.58 but =£0.62. 

To each of remaining flasks add 1 mL test culture suspension, 
4.042, as follows: Swirl flask, stopping just before suspension 
is added, to create enough centrifugal motion to prevent pooling 
of suspension at point of contact with test H 2 0. Add suspension 
midway between center and edge of liq. surface, immersing tip 
of pipet slightly below surface of H 2 0. Avoid touching pipet to 
neck or side of test flask during operation. 

From one of these 2 flasks transfer 1 mL aliquots to neutralizer 
blanks, 4.039(a), after intervals of 0.5, 1, 2, 3, 4, 5, and 10 min. 
Shake neutralizer blank thoroly immediately after adding sam- 
ple. Prep, serial diln plate counts from neutralizer blanks, using 
phosphate buffer diln H 2 0, 4.040(b), and subculture medium, 
4.037(a) for E. coli, and (b) for S. faecalis. 

After prepg diln plate counts, inoculate 5 lactose broth tubes, 
4.037(d), with 1.0 mL aliquots from each neutralizer blank tube 
for each time interval when E coli is used as the test organism, 
and 5 thioglycolate broth tubes, 4.037(c), with 1.0 mL aliquots 
from each neutralizer blank tube for each time interval when S. 
faecalis is test organism. 

Incubate all diln plates in inverted position and subculture 
tubes 48 hr at 37°. Use Quebec Colony Counting Chamber in 
reading diln plates and report results in terms of number of 
surviving bacteria/mL test H 2 0. Absence of colony growth on 
diln plates and absence of growth in all 5 lactose or thioglycolate 
tubes, as case may be, is necessary to show complete kill of test 
organism. 

Immediately after transferring 10 min interval sample from 
second flask to neutralizer blank tube, remove third flask from 
H 2 bath and det. residual available CI exactly as specified for 
first flask. Results should represent residual available CI present 
at 10 min exposure interval. To be acceptable, concn of available 



CI in this flask should be >0.4 ppm. Results in CI control test 
described above should show complete kill of E. coli and S. 
faecalis within 0.5 min. 

With unknown sample, prep. 2 flasks contg 199 mL each of 
soln at concn recommended or to be studied, using CI demand- 
free, unbuffered, freshly distd H 2 previously prepd in 1 L vol. 
flask where CI demand, as detd above, has been satisfied by 
addn of NaOCI soln. Place flasks in H 2 bath at 20 or 25°; let 
come to temp. Inoculate 1 flask with 1 mL std test culture 
suspension of E. coli and other with 1 mL std test culture 
suspension of 5. faecalis. Subculture at exactly same time 
intervals and in same manner used with NaOCI control except 
vary composition of neutralizer blank depending upon nature 
of chem. or mixt. of chems under investigation. For example, 
mixt. of Cl-contg chem. and quaternary NH 4 compd would 
require special neutralizer blank prepd by using both neutralizer 
stock solns, 4.038(a) and (b). 

Where no concn of chem. under study has been recommended 
and objective of study is to det. concn of unknown necessary 
to provide result equiv. to that obtained with CI control std, use 
series of three or four 500 mL flasks contg 199 mL of various 
concns of chem. and 1 mL stdzd culture suspension with each 
test organism. Report results as log (number of survivors) at 
each time interval both for CI controls and various concns of 
unknown under test. 

Lowest concn of unknown germicide or germicidal mixt. 
providing results equiv. to those obtained with NaOCI as CI std 
is considered lowest concn which could be expected to provide 
acceptable disinfecting activity in swimming pool water. 



TubercuSocidal Activity (9)— Official Final Action 

(Suitable for detg max. tuberculocidal diln of disinfectants used 
on inanimate surfaces) 

/. Presumptive In Vitro Screening Test Using 
Mycobacterium smegmatis 

4.045 Reagents 

(a) Test organism. — Mycobacterium smegmatis (PRD No. 1) 
(available from Microbiology Lab., Benefits and Field Services 
Div., EPA, Beltsville, MD 20705). Maintain on nutrient agar slants 
by monthly transfers. Incubate new stock transfer 2 days at 37°; 
then store at 2-5°. From stock culture inoculate tubes of Pros- 
kauer-Beck broth, (b)(7), incubate 48 hr in slanting position, 
carry 30 days, using 48 hr transfers, and use these 48 hr cultures 
to start test cultures. Inoculate 1 or 2 tubes of Proskauer-Beck 
broth. Incubate 6-7 days at 37°. Incubate tubes 48 hr in slanting 
position to provide max. surface aeration and then in upright 
position 4-5 days. Add 1.5 mL sterile 2.0% Bacto-Gelatin soln 
and homogenize culture with sterilized glass tissue grinder, 
4.016(e). Adjust to 20% T at 650 nm with sterile Proskauer-Beck 
broth for use in testing. 

( b) Culture media. — ( 7 ) Modified Proskauer-Beck broth. — Dis- 
solve 2.5 g KH 2 P0 4 , 5.0 g asparagine, 0.6 g MgS0 4 .7H 2 0, 2.5 g 
Mg citrate, 20.0 mL glycerol, 0.0046 g FeCI 3 , and 0.001 g 
ZnS0 4 .7H 2 in 1 L H 2 0. Adjust to pH 7.2-7.4 with 1/V NaOH. 
Filter thru paper, place 10 mL portions in sep. 20 x 150 mm 
tubes, and sterilize 20 min at 121°. Use for propagating 48 hr 
test starter cultures and 6-7 day test cultures. 

(2) Subculture media. — Use (7) with addn of suitable neutzg 
agents such as purified lecithin (Azolectin) or Na thioglycolate, 
where necessary. 

{3) Nutrient agar. — Prep, as in 4.001(c). Use to maintain stock 
culture. 

{4) Sterile distilled water.See 4.007(d). 



66 



4. Disinfectants 



AOAC Methods (1980) 



4.046 Apparatus 

(a) Glassware, water bath, transfer loops and needles, and 
petri dishes.— See 4.008(a), (b), (c), and <e). 

(b) Carriers.— See 4.016(g). 

4.047 Operating Technic 

Transfer 20 sterile Penicylinder carriers, using flamed ni- 
chrome wire hook, into 20 mL 6-7 day homogenized stdzd broth 
culture, 4.045(a), in sterile 25 x 150 mm medicanttube. After 15 
min contact, remove cylinders and place on end in vertical 
position in sterile petri dish matted with fitter paper, 4.008(e). 
Cover and place in incubator at 37° and let dry 3=20 min but ^60 
min. This will provide dried test carriers in groups of 20 in 
individual petri dishes. With each group of 20 carriers, add 1 
dried cylinder at 30 sec intervals to each of 20 tubes contg 10 
mL diln of germicide to be tested (at 20° in H 2 bath). Flame lips 
of medicant and subculture tubes. Immediately after placing 
carrier in medicant tube, swirl tube 3 times before placing it 
back in H 2 bath. (Thus, by time 20 tubes have been seeded, 9 
min and 30 sec have elapsed, leaving 30 sec interval prior to 
subculturing series at 10 min exposure for each carrier. The 30 
sec interval between transfers allows adequate time for flaming 
and cooling transfer hook and making transfer in manner so as 
to drain all excess medicant from carrier.) Transfer carrier to 10 
mL subculture media, 4.045(b)(2). Shake all subculture tubes 
thoroly and incubate 12 days at 37°. Report results as + (growth) 
or - {no growth). Where there is reason to suspect that results 
may be affected by bacteriostatic action of medicant carried 
over in subculture tubes, use suitable neutralizer in subculture 
media. 

Make s=30 carrier exposures at each of 3 relatively widely 
spaced dilns of germicide under test between no response and 
total response diln levels. Calc. % of carriers on which organism 
is killed at each diln. Using log.% probit paper (3 cycle logarithmic 
normal No. 32.376, Codex Book Co., Inc., Norwood, MA 02062), 
locate % kill points on diln lines employed (log scale). Draw 
best fitting straight line thru these 3 points and extend to 
intercept 99% kill line. Read diln line (log scale) at point of 
intercept This is presumed 95% confidence end point for 
product. (Do not use presumptive test organism for checking 
validity of this presumptive end point.) 

//. Confirmative In Vitro Test for Determining 
Tuber culocidal Activity 



4.048 



Reagents 



(a) Culture media. — (7) Modified Proskauer-Beck medium. — 
Prep, as in 4.045(b)(7), and in addn, place 20 mL portions in 25 
x 150 mm tubes. Use 10 mL portions for daily transfers of test 
cultures and 20 mL portions for subculturing porcelain cylinders. 

(2) Middlebrook 7H9 Broth Difco A— Dissolve 4.7 g in 900 mL 
H 2 contg 2 mL glycerol and 1 5.0 g agar. Heat to bp to dissolve 
completely. Distribute in 180 mL portions and autoclave 15 min 
at 121°. To each 180 mL sterile medium at 45°, add 20 mL 
Middlebrook ADC Enrichment (Difco) under aseptic conditions 
and distribute in 10 mL portions in sterile 20 x 150 mm tubes. 
Slant. Use to maintain test culture. 

(3) Middlebrook 7H9 Broth Difco B.— Dissolve 4.7 g in 900 mL 
H 2 contg 2 mL glycerol and 1.0 g agar. Heat to bp to dissolve 
completely. Distribute in 18 mL portions in 25 x 150 mm tubes, 
and autoclave 15 min at 121°. To each 18 mL sterile medium at 
45° add 2 mL Middlebrook ADC Enrichment under aseptic 
conditions. Use to subculture for survival. 

(4) Kirchners Medium Difco. — Dissolve 13.1 g in 1 L H 2 contg 
20 mL glycerol and heat to bp to dissolve completely. Distribute 



in 18 mL portions in 25 x 150 mm tubes and autoclave 15 min 
at 121°. To each 18 mL sterile medium at 50-55° add* 2 mL 
Middlebrook ADC Enrichment under aseptic conditions. Use to 
subculture for survival. 

(5) TB Broth Base Difco (without polysorbate 80). — Dissolve 
11.6 g in 1 L H 2 contg 50 mL glycerol and 1.0 g agar. Heat to 
bp to dissolve completely. Distribute in 18 mL portions in 25 x 
150 mm tubes,- and autoclave 15 min at 121°. To each 18 mL 
sterile medium at 50° add 2 mL Dubos Medium Serum (Difco) 
under aseptic conditions. Use to subculture for survival. 

(b) Test organism.— Mycobacterium bovis (BCG) (available 
from ITR Biomedical Research, University of Illinois Medical 
Center, 904 W Adams St, Chicago, IL 60607). Maintain stock 
cultures on culture medium (a){2) by monthly or 6 weeks 
transfer. Incubate new stock transfer 15-20 days at 37° until 
sufficient growth is indicated; then store at 2-5°. From stock 
culture, inoculate tube of culture medium (a)(7) and incubate 
21-25 days at 37°. Shake gently once daily for 9 days; then 
allow to remain quiescent until 21-25th day. Make daily transfers 
from 21 day cultures. Transfer culture to heat-sterilized glass 
tissue grinder, add 1.5 mL sterile 2% Bacto-gelatin soln (Difco), 
grind, and dil. with culture medium (a)(7) to give 20% T at 650 
nm. Use to inoculate porcelain cylinders used in test. Tests will 
be satisfactory only when organism is killed on all 10 carriers 
by aq. phenol (1+50) and shows survival after exposure to aq. 
phenol (1+75) control. Prep, dilns from 5% std phenol soln, 
4.002(f). 

(c) Sterile distilled water.^See 4.007(d). 

(d) Sterile normal horse serum without preservative.— Difco 
Laboratories or Microbiological Associates, 4733 Bethesda Ave, 
Bethesda, MD 20014. 

4.049 Apparatus 

(a) Glassware, water bath, transfer loops and needles, and 
petri dishes.^See 4.008(a), (b), (c), and (e). 

(b) Carriers.— See 4.016(g). 

4.050 Operating Technic 

Soak ring carriers overnight in 1/V NaOH; rinse with tap H 2 
and then with distd H 2 until distd H 2 is neut. to phthln; then 
rinse twice with distd H 2 0. Place clean ring carriers in multiple 
of 10 or 20 in capped erlenmeyer or 20 x 150 mm tubes. 
Autoclave 20 min at 121°, cool, and hold at room temp. Transfer 
10 sterile ring carriers, using flamed wire hook, into enough (ca 
15-20 mL) 21-25 day stdzd test culture, 4.048(b), in 25 x 150 
mm medication tube. After 15 min contact period, remove 
cylinders, using flamed wire hook, and place on end in vertical 
position in sterile petri dish matted with filter paper, 4.008(e). 
Cover, place in incubator at 37°, and let dry 5*20 min but =£60 
min. 

Let 10 tubes contg 10 mL use-diln germicide sample to be 
tested come to 20° in H 2 bath and add 1 contaminated cylinder 
carrier at either 30 sec or 1 min intervals to each tube. Imme- 
diately after placing carrier in medication tube, swirl 3 or 4 times 
before placing tube back in bath, (Thus, by time 10 tubes have 
been seeded, 9 min will have elapsed, plus 1 min interval before 
transfer of first carrier in series to individual tube of 10 mL 
serum, 4.048(d), or 10 mL neutralizer blank, 4.023(d), if 1 min 
intervals are used. This interval is constant for each tube with 
prescribed exposure period of 10 min. Interval between transfers 
allows adequate time for flaming and cooling wire hook and 
making transfer in manner so as to drain all excess medication 
from carrier.) Transfer carrier to 10 mL serum, 4.048(d), after 
exactly 1 min contact. Shake tube contg carrier in serum thoroly 
and place carrier in tube contg 20 mL broth, 4.048(a)(7). From 



AOAC Methods (1980) 



Laundry Additives 



67 



same tube, take 2 mL portions serum and place in any 2 of the 
subculture media, 4.048(a)(3), (4), (5). Repeat this with each of 
the 10 carriers. Incubate 1 tube of each subculture medium with 
2 mL sterile serum as control. Where there is reason to suspect 
that germicide to be tested may possess bacteriostatic action, 
use suitable neutralizer in lieu of serum. Shake each subculture 
tube thoroly, incubate 60 days at 37°, and report results as + 
(growth) or - (no growth). If no growth or only occasional 
growth is observed in subculture, incubate addnl 30 days before 
making final reading. Max. diln of germicide which kills test 
organism on the 10 carriers, and no growth in each of the 2 mL 
aliquots for 2 extra media, represents max. safe use-diln for 
practical tuberculocidal disinfection. 

Bacteriostatic Activity of Laundry Additives {10) 
Official First Action 

(Applicable to antimicrobial products, recommended for use 
during laundering operations, which are intended to provide 
residual bacteriostatic treatment to laundered fabric. Method 
includes treatment of fabric with product and subsequent bac- 
teriostatic testing of treated fabric.) 



Outer Ext. 



4.051 



Reagents 



(a) Culture media.— {1) Nutrient broth.— See 4.001(a). 

{2) Nutrient agar A. — See 4.001(c). Use for monthly transfer 
of stock cultures. 

(3) Nutrient agar B. — Boil 3 g beef ext, 5 g peptone (Anatone), 
8 g NaCI, and 10 g agar (Difco) in 1 L H 2 0. Transfer 100 mL 
portions to erlenmeyers, and autoclave 20 min at 121°. Use for 
agar plate tests to evaluate bacteriostatic activity of treated 
fabric. See also (c). 

(b) Test organisms. — Use Staphylococcus aureus ATCC No. 
6538 and Klebsiella pneumoniae, aberrant ATCC No. 4352 
(formerly Escherichia coli), and maintain as in 4.002(e). 

(c) 2,3,5-Triphenyi tetrazolium chloride.— Use as optional bi- 
ological indicator. With S. aureus, use 0.15% soln; with K. 
pneumoniae, aberrant, use 0.25% soln. Autoclave each 20 min 
at 121°. Apply as in 4.054. 

(d) Alkaline nonionic wetting agent — Prep. aq. soln contg 
0.5% alkyl phenol polyglycol ether wetting agent and 0.5% 
Na 2 C0 3 . Use to scour test fabric. 

4.052 Apparatus 

/(a) Test fabric. — 80 x 80 threads/ sq in. plain weave cotton 
print cloth, completely desized, bleached, and without bluing or 
optical brighteners (available from Test Fabrics, Inc., 55 Van 
Dam St, New York, NY 10013). Scour before use by boiling ca 
300 g 1 hr in 3 L H 2 contg 1 .5 g nonionic wetting agent and 1.5 
g Na 2 C0 3 . Then rinse fabric, first in boiling H 2 and then in cold 
H 2 0, until alt visual traces of wetting agent are removed. Air-dry 
and cut into long strip 5 cm (2") wide and weighing exactly 15 
9- 

(b) Stainless steel spindle. — Fabricate from single continuous 
piece of stainless steel wire 1 / i e" diam. and bent to contain 3 
horizontal extensions 5 cm (2") long connected by 2 vertical 
sections ca 5 cm (2") long. Shape so that vertical sections form 
150° angle, and sharpen free ends of 2 outer horizontal exten- 
sions to point {see Fig. 4:02). Use as carrier for test fabric. 
Primary objective of spindle is to prevent wadding or lodging 
of test fabric during agitation in exposures to test chem. solns. 

(c) Exposure chamber. — Clean, dry 1 pt Mason jar with rubber 
washer or gasket and metal screw cap. 

(d) Agitator. — Device to rotate Mason jar thru 360° vertical 
orbit of 10-20 cm (4—8") diam. at 45-60 rpm for 5 min. Launder- 
ometer or Tumble Jar described in AATCC70 B-1967, 43, B154, 
B155, or ASTM D583-63 is adequate. 



TOP 
VIEW 



Horizontal 
Extensions 



S> 



SIDE 
VIEW 



Horizontal 
Extensions 



Outer Ext. 



^>* -L 



*Points 
pencil 
sharp or 
canted 



FIG. 4:02 — Stain Bess steel spindle for winding test fabric 

(e) Water bath. — Thermostatically controlled at 25°. 

(f) Petri dishes.SterWe, 100 x 15 mm. 

(g) Glassware. — See 4.002(a). 

(h) Transfer loops and needles. — See 4.002(d). 



4.053 



Preparation of Fabric 



(a) Fabric mounting. — Pierce one end of prescoured, 1 5 g test 
fabric strip and secure onto an outer horizontal extension of test 
spindle; then wind strip around 3 horizontal extensions with 
enough tension to obtain 12 (but not 13) entire laps. Secure 
final end of test fabric strip to previous laps with stainless steel 
safety pin. 

(b) Fabric treatment with product. — DM. product as directed 
to 75 mL (most frequently, use directions are based on dry wt 
of laundry fabric equiv. to 15 g test fabric), add to Mason jar 
(exposure chamber), and maintain in H 2 bath at 25°. Add addnl 
materials to Mason jar as required by use directions for product. 
These are: 

( 7 ) Product recommended as final rinse additive in industrial 
laundering operation. — Add no addnl materials; 5:1 (v/w) treat- 
ment product soln to dry fabric ratio is representative of indus- 
trial laundering operations. 

(2) Product recommended as final rinse additive in home or 
coin-operated laundering operations. — Add 150 mL H 2 to Ma- 
son jar. Resultant 10:1 (v/w) treatment product soln to dry fabric 
ratio is representative of home and coin-operated laundering 
operations. 

(5) Product recommended as final rinse additive in both 
industrial and home laundering operations. — Prep. 2 jars contg 
product soln according to (7) and (2) so that 2 test fabric strips 
may be treated at different treatment product soln to dry fabric 
ratios (5:1 and 10:1 (v/w)). 

(4) Product recommended as final rinse additive and de- 
scribed as compatible with adjunct chemicals which may be 
used in this cycle {sours, bleaches, optical brighteners, softeners, 
etc).— Prep, so that required vol. of product treatment soln 
contains adjunct chemicals according to description and amts 
specified on product label or advertising literature. 

4.054 Operating Technic 

Place test spindle with test fabric in Mason jar contg product 
soln. Secure rubber gasket and Mason jar cap, remove from 
H 2 bath, place jar in agitator, and rotate 5 min. Addnl manip- 
ulation with test spindle is required if use directions do not 
specify addn of product in final rinse phase of laundry cycle. In 
this instance, to det. durability of antimicrobial agent in fabric, 
execute 3 rinse operations as follows: Immediately after end of 



68 



4. Disinfectants 



AOAC Methods (1980) 



initial 5 min agitation, drain treatment soln from Mason jar and 
replace with 100 mL H 2 0. Secure Mason jar contg test spindle, 
return to agitator, and rotate 2 min. Repeat operation twice 
more. 

Following all required fabric treatment operations, remove 
test spindle from Mason jar and unwind test fabric strip from 
spindle. Let test fabric strip air dry with long axis of strip in 
horizontal position. 

When test strip is dry, remove 1 sq in. bacteriological test 
samples. Five test samples are required for single bacteriostatic 
test against 1 test organism. In each such instance, at least 2 
test samples must be removed from middle 20% of length of 
test strip. 

Perform bacteriostatic agar plate tests as follows: Prep. 5 
replicate plates in each test for each organism. Sep. inoculate 
flasks contg 100 mL sterile, liq. (=^40°) nutrient agar B with 1 mL 
24 hr nutrient broth culture of 5. aureus and K. pneumoniae, 
aberrant. Immediately thereafter, if desired, add 1 mL appro- 
priate soln of 2,3,5-triphenyl tetrazolium chloride to inoculated 
nutrient agar B. Vigorously swirl contents of erlenmeyers to 
ensure complete mixing. Add 10 mL portions of inoculated agar 
to 100 mm sterile petri dishes, distribute evenly, and let cool 
and harden. As soon as plates harden, implant single 1 sq in. 
treated fabric test sample on center of 1 test agar plate surface. 
Using blunt forceps, press each fabric test sample onto agar 
surface to ensure complete and uniform contact. Incubate test 
plates 48 hr at 37°. If desired, test plates may be refrigerated 
18-20 hr before incubation. Following incubation, examine test 
plates to det. presence or absence of zones of inhibition along 
each side of test fabric sample. 



4.055 



interpretation 



Use clear zone of inhibition adjacent to each side of test fabric 
sample as index of bacteriostatic activity. Size of zone is not 



considered important, but zone is required to extend along 
entire edge to be acceptable. Score zone of inhibition along 
single side of sq test fabric samples as 1, so that for 5-replicate 
plate test, a score of 20 shows that bacteriostasis occurs along 
all 4 sides of each sample. Total score of 18/20 sides demon- 
strating bacteriostasis is required for effective demonstration of 
residual bacteriostatic activity of laundry fabric treated with 
antimicrobial laundry additive product during laundering op- 
eration. Unless qualified residual bacteriostatic claim is made, 
residual treatment must be bacteriostatic against both S. aureus 
and/C pneumoniae, aberrant. 

SELECTED REFERENCES 

(/) J. Roy. Sanit. Inst. 24, 424(1903); Am. J. Public Health 3, 
575(1913); U.S. Dept. Agr. Circ. 198 (1931); JAOAC 32, 
408(1949); 38, 465(1955); Soap Chem. Spec. 34, No. 10, 
79(1958). 

(2) J. Bacteriol. 49, 526(1945); Am. J. Vet. Res. 9, 104(1948); 
JAOAC 36, 466(1953). 

(3) Soap Sanit. Chem. 27, No. 2, 133(1951); JAOAC 38, 
274(1955); 40, 755(1957). 

(4) JAOAC 36, 480(1953); 39, 480(1956); 40, 759(1957); 49, 
721(1966). 

(5) Arch. Dermatol. Syphilol. 28, 15(1933); J. Bacteriol. 42, 
225(1941); 47, 102(1944); JAOAC 37, 616(1954); 38, 
274(1955). 

(6) Am. J. Public Health 38, 1405(1948); J. Milk Food Technol. 
19, 183(1956); Fed. Regist. 21, 7020(1956); JAOAC 41, 
541(1958). 

(7) JAOAC 44, 422(1961); 50, 763(1967); Soap Chem. Spec. 
38(2), 69(1962). 

{8) JAOAC 47, 540(1964); 48, 640(1965). 
{9) JAOAC 48, 635(1965); 50, 767(1967). 
[10) JAOAC 52, 836(1969). 



5* Hazardous Substances 



PAINT 
Lead (7) — Official Final Action 



5.001 



Reagents and Apparatus 



(a) Lead std solns.— {1) Stock soln.— *\ mg Pb/mL 1% HN0 3 . 
Dissolve 159.9 mg Pb(N0 3 ) 2 in HN0 3 (1+99) and dil. to 100 mL 
with HN0 3 (1+99). (2) Intermediate soln.— 300 ^g/mL dil. HN0 3 . 
Dil. 15 mL stock soln to 50 mL with 0.5 mL HN0 3 and H 2 0. (5) 
Working solns. — To each of seven 100 mL vol. flasks contg 1 mL 
HNO3, add resp. 0, 1, 2, 3, 4, 5, and 6 mL intermediate soln and 
dil. to vol. with H 2 (0, 3, 6, 9, 12, 15, and 18 fxg Pb/mL). 

(b) Atomic absorption spectrophotometer. — With Pb hollow 
cathode lamp and 4" single slot or 3 slot Boling burner head, 
capable of detecting 0.5 fxg Pb/mL, such as Perkin-Elmer Model 
403. Operating conditions: 283.3 nm, 0.7 nm band width slit, 
recorder response (if used) 0.25-1 sec time constant, air-C 2 H 2 
flame, with gas flows adjusted according to directions of man- 
ufacturer. 

(c) Heater for digestion. — Drill 7.5 cm Al block to hold ^16 
test tubes, 16 x 150 mm. Place on hot plate capable of main- 
taining medium at 160-170° (Corning PC 35, or equiv.). Sand 
bath may be used instead of Al block. 

(d) Boiling chips. — Unglazed boiling chips, 1.5 mm diam., Pb- 
free. 



5.002 



Determination of So/ids 



Thoroly mix samples manually for 10 min or mech. for 5 min. 
Accurately weigh 0.3-0.4 g into weighed Al dish, 63 mm diam. 
Add 3-5 mL hexane or pet ether to oil-based paints or H 2 to 
latex paints and swirl to disperse. Warm on hot plate while 
swirling until solv. has evapd and film is formed. Heat in oven 
4 hr at 105°, cool, and weigh. 

% Solids = g dried sample x 100/g sample. 



5.003 



Determination of Lead 



Introduce ca 0.6 g (0.3 mL) thoroly mixed sample near bottom 
of 16 x 150 mm test tube with syringe and weigh accurately. 
Add 5±0.2 mL HN0 3 and 2 boiling chips to each, including 
blanks. Place in block or bath at 90-100° so that liq. surface is 
slightly above heated surface. (Use hood.) After initial fuming 
has subsided, increase temp, until vapors are condensing in 
top 1-2 cm of tube (bath temp., 160-170°) and maintain at this 
temp. 3 hr. Cool to 50-60°, transfer to 25 mL vol. flask, including 
chips and any ppt, and rinse with four 4 mL portions H 2 0, 
transferring as much residue as possible. Dil. to vol. with H 2 
and let settle 0.5-1 hr. Floating residue may be removed by 
aspiration thru disposable pipet. 

Aspirate solns and stds into AA spectrophotometer, avoiding 
introduction of ppt. If A of sample is greater than highest std, 
dil. sample and re-aspirate. Det. /jlq Pb/mL from std curve. 
% Pb in paint solids = (jug Pb/mL) x F x 10~ 2 / 

(g sample x % solids in sample) 
F (diln factor) = 1/[(1/25) (b/c) id/e) . . J, 
where 25 = vol. original sample digest, b = aliquot of original 
25 mL dild to c mL; d ~ aliquot of c (mL) dild to e mL; etc. For 
dry paint films, % solids in sample = 100. 



PREPARATIONS CONTAINING FLUORIDES 

Fluoride (2) — Official Final Action 

5.004 Apparatus 

(a) pH meter. — With expanded mv scale (digital Model 110, 
Corning Scientific Instruments, Medfield, MA 02052, or equiv.), 
fluoride ion-selective electrode (Model 94-09, Orion Research 
Inc., 11 Blackstone St, Cambridge, MA 02139, or equiv.), and 
single junction ref. electrode, plastic sleeve-type (Model 90-01, 
Orion Research Inc., or equiv.). 

(b) Magnetic stirrer. — With Teflon-coated stirring bar. Use 
asbestos or foam mat to insulate sample from motor heat. 

(c) Beakers.— 4.5 02 (135 mL), polypropylene, or equiv. 

(d) Graph paper. — Linear or semi-antilog, vol. corrected No. 
90-00-90 Gran's plot paper (Orion Research Inc., or equiv.). 



5.005 Reagents 

(a) Buffer soln.— pH 6.0. Add 77.0 g NH 4 OAc and 0.452 g NH 4 
citrate to 1 L H 2 0. Adjust to pH 6.0 with HOAc. 

(b) Fluoride std sofn.—l mg F/mL. Prep. 2.2108 g NaF 
(reagent grade, dried 4 hr at 105°)/L buffer soln. {Caution: See 
51.084.) Store in leakproof plastic bottles. Compare with 1 mg 
F/mL soln prepd from USP Ref. Std; equiv. reading of ±1 mv 
is satisfactory. 



5 .006 Determination 

{Stir all solns constantly at same rate thruout titrns. Let electrodes 

equilibrate s=2 min before addn of F std soln and 30 sec after 

each addn of F std soln.) 

(a) Blank. — Record mv values (£') of 100 mL buffer soln after 
addn of 4 mL std F soln from 10 mL buret and after each addnl 
mL up to 10 mL. (Preliminary mv values will not fall on linear 
range of response curve.) Vol. std soln added = V. 

(b) Samples. — Est. molarity of samples from direct reading. 
Dil. samples, if necessary, to ca 0.00 W F. Transfer 50 mL sample 
soln to beaker and add 50 mL buffer soln. Record initial mv 
reading, using expanded scale {£„). If initial reading is <-50 
mv, soln is too coned. Dil. sample to avoid asymptotic slope. 
Record mv values {£) after each mL F std soln is added up to 10 
mL. Rinse electrodes with H 2 between samples. Vol. std soln 
added = V. 



5.007 



Calculations 



(a) Linear graph paper. — For each addn of F std soln and 
corresponding E value, calc. for blank: 

Z' = antilog [log(V + V') - 0.017(F)], 
where V is original vol. soln to which F std soln was added 
(100mL) and £' is treated algebraically (+ or - as read). Plot Z' 
against mL (mg) F std soln added and extrapolate to intersection 
of mL (mg) F axis to obtain mL (mg) F in blank, l/ e '. In graph, 
assign horizontal axis to mL (mg) F, with at center and mL 
(mg) F increasing in both directions to left and right. Assign Z 
values to vertical axis. Plot actual readings of mL (mg) F on 
right portion of horizontal axis so that extrapolation will fall on 
left portion of axis. 



69 



70 



5. Hazardous Substances 



AOAC Methods (1980) 



Similarly, for original readings and each addn of F std soln 
and corresponding E value, calc. for sample: 

Z - antilog [log(l/ + V) - 0.017(E)], 
where V Q is original vol. soln to which F std soln was added (100 
mL). Plot 7 against mL (mg) F std soln on same graph as blank 
and extrapolate to intersection of mL (mg) F axis to obtain mL 
(mg) F in sample, V e . 

(b) Semi-antilog paper. — Plot E directly for both blank and 
sample, descending 5 mv for each major line crossing vertical 
axis. At top of vertical axis place most neg. E reading which still 
allows extrapolation of V e on left portion of mL (mg) side of 
horizontal axis. Obtain V e and l/ e ' by extrapolation to left side 
of mL (mg) F. 

% f = (l/ e - f e ') x (B x 100)/[W x C x 1000 (mg/g)], 
where B = vol. of diln, W = mL or g sample, and C - aliquot 
(50 mL max.) buffered to 100 mL. 



PREPARATIONS CONTAINING METHANOL 

Methanol (3) — Official Final Action 

(Applicable in presence of acetone, BuOAc, EtOH, isopropanol, 

hexane, MeEt ketone, CH 2 CI 2 , Me Cellosolve, paraffin, toluene, 

and H 2 0. This includes many paint removers, fuels, liq. sanders, 

antifreezes, and paint products.) 

5.008 Apparatus and Reagents 

(a) Gas chromatograph. — With flame ionization detector and 
oven capable of temp, changes 5=5°/min near 160° or preferably 
temp, programmer. Column.^- 1.8 m (6') x 4 mm id packed with 
120-150 mesh Porapak R (Waters Associates, Inc., PO Box 246, 
Milford, MA 01757); condition 2 hr at 235°. Conditions: Temps 
(°): injection ca 200, column ca 160, detector ca 210; N flow ca 
25 mL/min; set electrometer so that 8 /aL std soln provides at 
least half scale peak. Adjust column temp, and N flow so that 
MeOH retention time is ca 5-7 min. 

(b) Methanol std soln.— 0.4% (v/v). Dil. 4.00 mL MeOH to 100 
mL with dioxane; dil. 10.0 mL of this soln to 100 mL with 
dioxane. Rinse pipet into flask before dilg to vol, with dioxane. 
Prep, fresh daily. 



5.009 



Preparation of Sample 



(a) For asphalt-base tar compounds and viscous adhesives. — 
Refrigerate unopened sample container ^3 hr (longer for larger 
containers) at 1-10°, open container, and mix well; close con- 
tainer and refrigerate 30 min more. Transfer 1.5-3 g sample to 
tared, 250 mL, wide-mouth g-s erlenmeyer (tared with stopper 
in place). Let sample reach room temp, in stoppered erlenmeyer 
and weigh. Refrigerate 30 min and quickly add 1 00.0 mL dioxane. 
Stopper and shake mech. 1 hr. Refrigerate 30 min and filter thru 
rapid paper (S&S sharkskin, or equiv.). Filter as quickly as 
possible, covering funnel with watch glass and placing funnel 
against neck of narrow-mouth g-s receiver. Proceed as in 5.010, 
dilg with dioxane, if necessary. 

(b) For other less viscous products. — Prep, soln with pi pets 
and vol. flasks to contain ca 0.4% (v/v) MeOH, dilg with dioxane. 
Avoid excessive shaking of semiviscous products and do not fill 
pipet above mark. (Use safety pipet filler to draw liq. to mark 
and hold until transfer.) Wash pipet with dioxane and add 
washings to soln. 

If MeOH concn is unknown, prep. 2% soln. Prep, addnl dilns 
as needed. 



5.010 Determination 

Inject portion std soln with 10 pi syringe. Note vol. At R MA 
(retention time relative to MeOH) ca 0.5, inject portion sample 
soln. Note vol. At R MA ca 2 (from second injection), repeat 
injection of std soln. At R MA ca 0.5 (from third injection), repeat 
injection of sample soln. After MeOH from fourth injection 
elutes, increase column temp, to 235° as rapidly as possible for 
time ca 4 x /? M " A until all dioxane (R MA ca 5) is removed from 
column. Cool column to 160° and repeat sequence for subse- 
quent sample. Modify injection time if necessary to sep. MeOH 
from other peaks. (Note: Injection sequence is used only to save 
time; it need not be used if desired.) 



5.011 



Calculation 



Det. retention areas for each MeOH peak by multiplying peak 
ht by retention distance. Average retention areas for sample 
(RA) and for std (RA'). Presence of solv. in column changes 
retention times, requiring use of retention areas in calcn. 

% MeOH (w/v) in sample soln - F x (RA/RA') 

x (V/V) x C x 0.79, 
where F = diln factor, C = % (v/v) std soln, V and V = vol. 
sample and std soln injected, resp., and 0.79 = density of MeOH, 



Volatile Denatu rants in Alcoholic Products (4) 
Official First Action 

5.012 Apparatus and Reagents 

(a) Gas chromatograph and integrator. — See 19.001(a) and 
(b). 

(b) Std solns.—6% (v/v). Dil. 6.00 mL of each denaturant of 
interest to 100 mL with anhyd. alcohol in sep. vol. flasks. Approx. 
slopes and retention times relative to n-PrOH are given in Table 
5:01. 

Table 5:01 Approximate Slopes and Retention Times Relative 
to /f-Propyl Alcohol (RT) for Denaturants 



Compound 


Slope 


RT 


Acetone 


0.207 


0.694 


Benzene 


0.464 


2.309 


/7-Butyl alcohol 


0.269 


2.283 


sec-Butyl alcohol 


0.246 


1.621 


Chloroform 


0.058 


1.543 


Ethyl acetate 


0.192 


1.640 


Ethylene glycol monoethyl ether 


0.187 


3.868 


Ethylene glycol monomethyl ether 


0.151 


2.071 


Isopropanol 


0.210 


0.727 


Methanol 


0.130 


0.266 


Methyl isobutyl ketone 


0.275 


5.436 


Toluene 


0.454 


5.302 



5.01 3 Determination 

Pipet 25 mL of each expected denaturant std soln into sep. 
flasks and add 1.00 mL n-PrOH as internal std. Proceed as in 
19.002, starting with "Cap immediately . . .", except inject 0.3 
fil portions. Det. peak areas and calc. slope for each compd as: 

S x - (PA x /PA t )/6.0Q, 
where PA* and PA X = peak areas of compd X in std soln and of 
Aj-PrOH internal std, resp., and 6.00 = % compd X in std soln. 
Slopes and retention times should approximate those of Table 
5:01. 

% Compd X in sample = (PA/PA) = (1/S X ), 
where PA = peak area of compd X in sample. 



AOAC Methods (1980) 



Selected References 



71 



PREPARATIONS CONTAINING PHENOL 

Phenol 

Method I (5)— Official Final Action 

(Applicable to com. cresols, saponified cresol solns, coal tar 

dips, and disinfectants, and to kerosene solns of phenols in 

absence of salicylates or /3-naphthol) 

5.014 Reagents 
{Caution: See 51.026 and 51.065.) 

(a) Dilute nitric acid. — Aerate HN0 3 until colorless and dil. 1 
vol. with 4 vols H 2 0. 

(b) Mill on reagent — To 2 mL Hg in 200 mL erlenmeyer under 
hood, add 20 mL HN0 3 . After first violent reaction, shake as 
needed to disperse Hg and maintain action. After ca 10 min, 
when action practically ceases even in presence of undissolved 
Hg, add 35 mL H 2 0, and if basic salt seps, add enough dil. HN0 3 
to dissolve it. Add 10% NaOH soln dropwise with thoro mixing 
until curdy ppt that forms after adding each drop no longer 
redissolves but disperses as permanent turbidity. Add 5 mL dil. 
HN0 3 and mix well. Prep, fresh daily. Millon reagent is danger- 
ously poisonous and should not be transferred with ordinary 
pipet and mouth suction unless protective trap is used. 

(c) Phenol std soln. — Dissolve weighed amt pure phenol 
(congealing point 3=40°) in enough H 2 to make 5*1% soln. On 
day it is to be used, dil. to make 0.025% aq. soln (final std). 

(d) Formaldehyde soln.— D\l 2 mL 37% HCHO soln to 100 mL 
with H 2 0. 

(e) Methyl orange indicator. — 0.5% aq. soln. 

5.015 Apparatus 

(a) Nessler cylinders. — 50 mL tall-form, matched. 

(b) Test tubes.— Approx. 180 x 20 mm, with rubber stoppers, 
marked at 25 mL. 

(c) Water bath for heating test tubes. — Beaker contg disk of 
wire gauze raised ca 2.5 cm from bottom may be used. 

5.01 6 Preparation of Sample 

(a) Commercial creso/. — Weigh by difference ca 2.5 g sample 
into 250 mL vol. flask, dissolve in 10 mL 10% NaOH soln, and 
dil. to vol. with H 2 0. 

(b) Saponified cresol solns, coal tar dips and disinfectants, 
kerosene solns of phenols, etc. — Weigh by difference ca 5 g 
sample (or use 5 mL and calc. wt from density) into 250 mL vol. 
flask and dil. to vol. with H 2 0. With products consisting largely 
of kerosene, bring H 2 level to mark and take aliquots from aq. 
portion only. 



5.017 



Determination 



Transfer 5 mL aliquot prepd soln to 200 mL vol. flask and 
promptly dil. to ca 50 mL. Add 1 drop Me orange, (e), and then 
dil. HNO3 until soln is practically neut. Dil. to vol. and shake 
well. 

Place 5 mL dild soln in each of 2 marked test tubes; in each 
of 2 addnl test tubes place 5 mL std phenol soln. Flow 5 mL 
Millon reagent down side of each tube, mix, and place tubes in 
boiling H 2 bath; continue boiling exactly 30 min, cool imme- 
diately and thoroly by immersion in bath of cold H 2 **10 min, 
and add 5 mL dil. HN0 3 to each tube. 

Mix well and add 3 mL HCHO soln to one of each pair of 
tubes. Dil. all tubes to 25 mL mark with H 2 0, stopper, shake 
well, and let stand overnight. (Tubes contg HCHO fade to yellow; 
others show orange or red color.) 



Pipet 20 mL from each of the 2 phenol tubes to 100 mL vol. 
flasks; add 5 mL dil. HN0 3 to each, dil. to vol., and mix. (Red 
flask contains "phenol std/' yellow flask "phenol blank.") Trans- 
fer these solns to burets. Pipet 10 mL of each sample soln into 
Nessler tubes. {The orange or red constitutes the "unknown" 
and the yellow the "sample blank." Mark each Nessler tube 
distinctly to avoid confusion.) To "sample blank" tube add 
measured amt of "phenol std" and add same vol. "phenol 
blank" to "unknown." Agitate thoroly (aided by insertion of 
rubber stoppers, if necessary), and compare colors. When tubes 
are brought to match, each mL phenol std used = 1% phenol 
if sample weighing exactly 5 g was used, or 2% if exactly 2.5 g 
was used. 

Note. — Take following precautions: Pair of phenol tubes pro- 
vides enough final solns to assay several unknowns, but all the 
latter must have accompanied phenol solns thruout entire 
process with identical reagents and treatment. If end point is 
inadvertently overrun it is possible to work back to it, but since 
mistakes may be made in this operation it is better to repeat 
comparison on fresh portions from original tubes. Too much 
delay in matching tubes must be avoided after titrn is started, 
otherwise excess HCHO present in blanks may have time after 
mixing to affect intensity of red color. 

5.018 Method II [6)— Official Final Action 

(Applicable to detn of phenol in presence of salicylates) 

Weigh by difference 10 g sample into separator (or use 10 mL 
and calc. wt from density of sample). Add 50 mL kerosene and 
ext with three 100 mL portions H 2 0. Filter aq. exts thru wet filter 
into 500 mL vol. flask, dil. to vol. with H 2 0, and proceed as in 
5.017. 

When tubes are brought to match, each mL phenol std used 
= 1% phenol if sample weighing exactly 10 g was used. 

SODA LYE 
Carbonate and Hydroxide (7) — Official Final Action 

5.01 9 Determination 

Weigh ca 10 g sample from weighing bottle, dissolve in C0 2 - 
free H 2 0, and dil. to definite vol. Titr. aliquot with 0.5/V HCI, 
50.011 — 50.012, using Me orange, 5.014(e), and note total alky 
found. Transfer equal aliquot to vol. flask and add enough 10% 
BaCI 2 soln to ppt all carbonate, avoiding any unnecessary excess. 
Dil. to vol. with C0 2 -free H 2 0, stopper, shake, and let stand. 
When liq. clears, pipet off one-half and titr. with the 0.5/V HCI, 
using phthln; mL 0.5/V acid required for this titrn x2 = mL 0.5/V 
acid equiv. to NaOH present in original aliquot. Difference 
between this figure and mL 0.5/V HCI required for total alky - 
mL 0.5/V acid equiv. to Na 2 C0 3 present in aliquot. Calc. % Na 2 C0 3 
and NaOH. 

EARTHENWARE 

5.020 Cadmium and Lead 
See 25.031—25.037. 

SELECTED REFERENCES 

(7) JAOAC57, 614(1974). 
{2) JAOAC 56, 798(1973). 

(3) JAOAC 54, 558(1971); 55, 242(1972). 

(4) JAOAC 57, 148(1974). 

(5) USDA Bull. 1308, p. 17; JAOAC 13, 160(1930). 

(6) Ind. Eng. Chem., Anal. Ed. 1, 232(1929). 

(7) Sutton "Systematic Handbook of Volumetric Analysis," 10th 
ed., p. 61(1911). 



6. Pesticide Formulations 



(Pesticide ref. stds may be available from the following: Alltech 
Associates, 202 Campus Dr, Arlington Hts, IL 60004; Analabs, 
Inc.; Applied Science Laboratories; Chem Service Inc., West- 
chester, PA 19380; ICN-K&K Laboratories, Inc.; RFR Corp., 1 
Main St, Hope, Rl 02831; and Supelco.) 



GENERAL METHODS 

6.001 Sampling — Procedure 

{Caution: See 51.041.) 

Examine shipping cases closely for code numbers, different 
labels, and other pertinent information. Give special attention 
to products subject to deterioration. 

Caution; Use care in sampling and transporting toxic materials 
to avoid personal injury and contamination of transportation 
facilities in case of breakage. When dealing with rodenticides 
and weed-killers, avoid mutual contamination with other prod- 
ucts during transportation. 

Mark each sample container according to laboratory require- 
ments. 

(a) Smaii package retail units. — Take one unopened unit (1 lb 
if dry, 1 pt if liq.), except take min. of 2 units of small baits in 
cake form. Size of sample is governed by composition of material 
and anal, methods. 

(b) Large package dry products {25 lb or more). — Sample 
unopened containers, using trier long enough to reach bottom 
of container by inserting into container at one edge or corner 
and probing diagonally toward opposite edge or corner. Take 
cores by code or batch number. Analyze cores from same code 
or batch number as composite or individually. Clean trier thoroly 
after sampling each batch. 

Store samples in air-tight glass, metal, plastic, or cardboard 
containers. 

(c) Large package liquid products (5 gallons or more). — Use 
glass, plastic tubing, or stainless steel trier with plunger, or 
rubber tubing for certain materials. Store samples in glass or 
containers of other noncorrosive material with screw top caps 
lined with Teflon or other inert material. Plastic containers may 
be used only for carefully selected products. 

6.002 Sampling of Pressurized Containers (1) 
Official First Action 

Delivery assembly. — Construct U-shaped stainless steel as- 
sembly from %" od tubing, with 1 arm 70 mm long with front 
and back ferrules {Swagelok No. SS-204-1 and SS-203-1, 
Crawford Fitting Co., 29500 Solon Rd, Solon, OH 44139) per- 
manently swaged 3 mm from end, and other arm 135 mm 
longer (205 mm total length) sepd by 75 mm bend. Remove 
spray head from can. For male-type outlet, attach assembly 
from above ferrule to outlet with short piece of Tygon tubing. 
For female-type outlet, insert ferrule directly into outlet. Sample 
is released from can by carefully applying firm hand pressure 
to shoulder of assembly. See Fig. 6:01. 

Sampling. — Remove cover and spray head from can. Place 
can and delivery assembly in 800 ml beaker and weigh (W ). 
Shake can vigorously 5 min. Immediately after shaking attach 
delivery assembly to spray outlet. Immerse long end into 250 
mL separator contg 100 mL CHCI 3 . Press shoulder to deliver ca 
9 g sample (ca 10 sec) into CHCI 3 . Return can and delivery 
assembly to beaker and weigh. Shake can vigorously 3 min and 
repeat delivery of sample until total of 10-15 g is obtained in 



CHCI 3 . Reweigh can and delivery assembly in beaker (IV). Calc. 
sample wt as {W - W). 

6.003 Preparation of Sample — Official Final Action 

Thoroly mix all samples before analysis. Det. H 2 0-sol. As on 
samples as received, without further pulverization or drying. In 
case of lye, NaCN, or KCN, weigh large amts in weighing bottles 
and analyze aliquots of their aq. solns. 

6.004 Moisture — Official Final Action 

{Applicable to Paris green, powd Pb arsenate, Ca arsenate, Mg 

arsenate, Zn arsenite, powd Bordeaux mixt., and Bordeaux mixt. 

with arsenicals) 

Dry 2 g to const wt at 105-110° and report loss in wt as 
moisture. 



ARSENIC 

Total Arsenic — Official Final Action 

Hydrazine Sulfate Distillation Method (2) 

{Nitrates do not interfere. Applicable to detn of total As in Paris 

green, Pb arsenate, Ca arsenate, Zn arsenite, Mg arsenate, and 

Bordeaux mixt. with arsenicals) 



6.005 

{a) Arsenious 
50.005-50.006. 



oxide std soln. — 0.1 or 



PRESS 



Reagents 

0.05/V. See 




FIG. 6:01 — Delivery assembly for sampling pressurized containers 



72 



AOAC Methods (1980) 



Arsenic 



73 



(b) Iodine std soln.—OA or 0.05/V. See 50.018-50.019. 

(c) Bromate std sol n. — 0.1 or 0.05/V. Dissolve ca 2.8 or 1.4 g 
KBr0 3 in boiled H 2 and dil. to 1 L Stdze as follows: Pipet 25 
mL aliquots As 2 3 soln, (a), into 500 mL erlenmeyers. Add 15 
mL HCI, dil. to 100 mL, heat to 90°, and titr. with the KBr0 3 soln, 
using 10 drops Me orange, (g). Do not add indicator until near 
end of titrn, and agitate soln continuously to avoid local excess 
of KBr0 3 soln. Add KBr0 3 soln very slowly near end point; at 
end point soln changes from red to colorless. 

(d) Hydrazine sulfate-sodium bromide soln. — Dissolve 20 g 
N 2 H 4 .H 2 S0 4 and 20 g NaBr in 1 L HCI {1 +4). 

(e) Sodium hydroxide soln. — Dissolve 400 g NaOH in H 2 and 
dil. to 1 L 

(f) Starch indicator. — Mix ca 2 g finely powd. potato starch 
with cold H 2 to thin paste; add ca 200 mL boiling H 2 0, stirring 
constantly, and immediately discontinue heating. Add ca 1 mL 
Hg, shake, and let soln stand over the Hg. 

(g) Methyl orange indicator. — 0.05%. Dissolve 0.5 g Me or- 
ange in H 2 and dil. to 1 L 

6.006 Apparatus 

See Fig. 6:02. Set 500 mL distn flask on metal gauze that fits 
over circular hole in heavy sheet of asbestos board, which in 
turn extends out far enough to protect sides of flask from direct 
flame of burner. First receiving flask holds 500 mL and contains 
40 mL H 2 0; second holds 500 mL and contains 100 mL H 2 0. Vol. 
in first flask should be =£40 mL, otherwise compd of As may 
sep. that is difficult to dissolve without danger of loss of AsCI 3 . 
Keep both flasks cool by placing in pan of circulating H 2 0, or 
contg H 2 and ice. 



6.007 



Determination 



(Caution: See 51.041 and 51.078.) 

Weigh sample contg =£0.4 g As and transfer to distg flask. 
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, close flask with stopper that 
carries funnel tube, and connect side tube with condenser. Boil 
2-3 min, add 100 mL HCI from dropping funnel, and distil until 
vol. in distg flask is reduced to ca 40 mL; add 50 mL more HCI 
and continue distn until vol. is again reduced to ca 40 mL. Wash 
down condenser, transfer contents of receiving flasks to 1 L vol. 
flask, dil. to vol., mix thoroly, and proceed as in (a) or (b): 



(a) Pipet 200 mL aliquot into erlenmeyer and nearly neutze 
with NaOH soln, using few drops phthln, and keeping soln well 
cooled. If neut. point is passed, add HCI until again slightly acid. 
Neutze with NaHC0 3 , add 4-5 g excess, and add std I soln from 
buret, shaking flask continuously until yellow color disappears 
slowly from soln. Add 5 mL starch indicator and keep adding 
std I soln dropwise to permanent blue. 

(b) Pipet 200 mL aliquot into erlenmeyer and titr. with KBr0 3 
soln, (c), beginning ". . . heat to 90° . . ." 

Calc. % As. Report as As 2 3 or As 2 O s , according to whether 
As is present in trivalent or pentavalent form. If condition of 
arsenic is unknown, report as As. 

iodimetric Method (5) 

(Applicable in presence of sulfides, sulfites, thiosulfates, and 
large amts of S or org. matter) 



6.008 



Reagent 



Sodium thiosulfate soln. — Dissolve 13 g crystd Na 2 S 2 3 .5H 2 
in H 2 and dil. to 1 L 
See 6.005 for other reagents and solns and 6.006 for app. 



6.009 



Determination 



[Caution: See 51.041 and 51.078.) 

Weigh sample contg =e0.4 g As and transfer to distg flask. 
Add 50 mL N 2 H 4 .H 2 S0 4 -NaBr soln, 6.005(d), and distil as in 
6.007. Dil. distillate to vol. in 1 L vol. flask, mix thoroly, and 
transfer 200 mL aliquot to 400 mL Pyrex beaker or porcelain 
casserole. Add 10 mL HN0 3 and 5 mL H 2 S0 4 , evap. to sirupy 
consistency on steam bath, and then heat on hot plate to white 
fumes of H 2 S0 4 . Cool, and wash into 500 mL erlenmeyer. If vol. 
H 2 S0 4 is appreciably lessened by fuming, add enough H 2 S0 4 to 
make total vol. ca 5 mL Dil. to 100-150 mL, add 1.5 g Kl, and 
boil until vol. is reduced to ca 40 mL. Cool under running H 2 0, 
dil. to 100-150 mL, and add Na 2 S 2 3 soln, 6.008, dropwise until 
I color just disappears. Nearly neutze H 2 S0 4 with NaOH soln, 
6.005(e), finish neutzn with NaHC0 3 , add 4—5 g excess, and titr. 
with std I soln as in 6.007(a). From mL std soln used, calc. % As 
in sample. Report as As 2 3 , As 2 5 , or As as in 6.007. 




FIG. 6:02 — Apparatus for distilling arsenious chloride 



74 



6. Pesticide Formulations 



AOAC Methods (1980) 



Ion Exchange Method [4) — Official Final Action 

(Applicable to inorg. arsenates and arsenites) 

6.010 Apparatus 

Ion exchange column. — Use All inn filter tube 10 x 2.7 cm od 
with coarse filter disk. Attach piece of rubber tubing to bottom 
of filter tube and regulate flow with Hoffman clamp. To tube 
add aq. slurry of Dowex 50W-X8, 50-100 mesh, using resin bed 
vol. of 12 ml_, and place 500 ml_ separator above tube. 

Regenerate resin bed before each run by first back-washing 
column few min with H 2 0; then elute with 350 mL 2/V HCI 
followed by 200 mL H 2 at 20 mL/min. 

6.01 1 Preparation of Sample 

{Caution: See 51.041.) 

Weigh 200 mg sample (100 mg if As content is >30%) into 
150 mL beaker, add 7 mL HN0 3 , and bring to bp. Add 3 mL 2/V 
KBr0 3 and evap. to dryness, avoiding spattering. Backwash and 
regenerate resin during this evapn. Dissolve cooled residue in 
2 mL 6/V HCI without heat and add 8 mL H 2 0. Filter into separator, 
and wash filter with three 10 mL portions H 2 0. (If residue 
dissolves completely in 2 mL 6/V HCI, omit filtration, and dil. 
directly to 40 mL.) Pass soln thru resin column at 20 mL/min 
and collect eluate in 250 mL erlenmeyer. Wash separator and 
column with 20 and 40 mL portions H 2 into same erlenmeyer. 



6.012 



Determination 



Add 50 mL HCI to eluate to make 4/V. Add 1 g NaHC0 3 , 0.2 g 
at time, swirling constantly. Add 1 g Kl, stopper, and swirl until 
all Kl dissolves. After 5 min, titr., without starch indicator, with 
0.05/V Na 2 S 2 3 , 50.037—50.038, to disappearance of I. (Recog- 
nition of end point is facilitated by titrg on porcelain stand. In 
presence of starch, reaction between I and Na 2 S 2 3 is retarded, 
so appreciable amt of Na 2 S 2 3 reacts with acid. End point 
becomes indistinct if >30 mL Na 2 S 2 3 is used in titrn.) 1 mL 
0.05/V Na 2 S 2 3 = 1.873 mg As. 



Water-Soluble Arsenic — Official Final Action 

(Applicable to detn of H 2 0-sol. arsenic in Pb arsenate, Ca 

arsenate, Zn arsenite, Mg arsenate, and Bordeaux mixt. with 

arsentcals) 



6.013 



Determination 



{Caution: See 51.041.) 

To 2 g original sample if powder, or4g if paste, in 1 L Florence 
flask, add 1 L recently boiled H 2 that has been cooled to 32°. 
Stopper flask and place in constant temp. H 2 bath at 32°. Digest 
24 hr, shaking hourly 8 hr during this period. Filter thru dry 
filter, tf filtrate is not clear, refilter thru buchner contg paper and 
enough Filter-Cel coating to give clear soln. Discard first 50 mL. 

Transfer 250-500 mL clear filtrate to erlenmeyer, add 3 mL 
H 2 S0 4 , and evap. to ca 100 mL on hot plate. Add 1 g Kl, and 
continue boiling until vol. is ca 40 mL. Cool, dil. to ca 200 mL, 
and add Na 2 S 2 3 soln, 6.008, dropwise, until I color is exactly 
removed. (Avoid use of starch indicator at this point.) Neutze 
with NaHC0 3 , add 4-5 g excess, titr. with std I soln, shaking 
flask continuously, until yellow disappears slowly, add 5 mL 
starch indicator, 6.005(f), and continue titrn to permanent blue. 
Correct for amt std I soln necessary to produce same color, 
using same reagents and vol. From mL std I soln used, calc. 
% H 2 0-sol. As in sample. 



LEAD (5)— OFFICIAL FINAL ACTION 

(Applicable to such prepns as Bordeaux-Pb arsenate, Bordeaux- 
Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca arsenate) 

{Caution: See 51.026, 51.031, 51.041, 51.059, and 51.078.) 



6.014 



Determination 



Weigh 1 g powd sample and transfer to beaker. Add 5 rwLHBr 
(ca 1.38 sp gr) and 15 mL HCI, and evap. to dryness to remove 
As. Repeat treatment; add 20 mL HCI, and again evap. to 
dryness. Add 25 mL 2/V HCI to residue, heat to bp, filter 
immediately to remove Si0 2 , and wash with boiling H 2 to vol. 
of 125 mL. See that all PbCI 2 is in soln before filtering; if it will 
not dissolve completely in 25 mL 2/V acid, add 25 mL more and 
dil. filtrate to 250 mL. Pass in H 2 S until pptn is complete. Filter, 
and wash ppt thoroly with 0.5/V HCI satd with H 2 S. Save filtrate 
and washings for Zn detn. 

Transfer paper with sulfides of Pb and Cu to 400 mL Pyrex 
beaker and completely oxidize all org. matter by heating on 
steam bath with 4 mL H 2 S0 4 and ca 20 mL fuming HN0 3 in 
covered beaker. Evap. on steam bath, and then completely 
remove HN0 3 by heating on hot plate to copious white fumes 
of H 2 S0 4 . Cool, add 2-3 mL H 2 0, and again heat to fuming. Cool, 
add 50 mL H 2 and 100 mL alcohol, and let stand several hr 
(preferably overnight). Filter thru gooch, previously washed with 
H 2 0, then with acidified alcohol { 100 parts H 2 0, 200 parts alcohol, 
and 3 parts H 2 S0 4 ), and finally with alcohol, and dried at 200°. 
Wash ppt of PbS0 4 in crucible ca 10 times with acidified alcohol, 
and then with alcohol, to remove H 2 S0 4 . Retain filtrate and 
washings for Cu detn, if desired. 

Dry at 200° to const wt, keeping crucible covered to prevent 
loss from spattering. From wt PbS0 4 , calc. % Pb in sample, 
using factor 0.6832. 



COPPER (5)— OFFICIAL FINAL ACTION 

(Applicable to such prepns as Bordeaux-Pb arsenate, Bordeaux- 
Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca arsenate) 

6.015 Electrolytic Method 

Evap. filtrate and washings from PbS0 4 pptn, 6.014, to fuming; 
add few mL fuming HNO z to destroy org. matter, and continue 
evapn to ca 3 mL. Take up with ca 150 mL H 2 0, add 5 mL HN0 3 , 
and filter if necessary. Wash into 250 mL beaker, adjust vol. to 
200 mL, and electrolyze, using rotating anode and weighed 
gauze cathode with current of 2-3 amp. After all Cu has 
apparently deposited (ca 30 min), add 15-20 mL H 2 to electro- 
lyte and continue electrolysis few min. If no further deposition 
occurs on newly exposed surface of electrode, wash with H 2 
without breaking current either by siphoning or quickly replacing 
beaker with electrolyte successively with 2 beakers of H 2 0. 
Interrupt current, rinse cathode with alcohol, dry few moments 
in oven, and weigh. Calc. % Cu in sample. 

6.016 Volumetric Thiosulfate Method 

Proceed as in 6.015 to point at which filtrate and washings 
from PbS0 4 pptn are treated with fuming HN0 3 and evapd to 
vol. of ca 3 mL. Take up in ca 50 mL H 2 0, add NH 4 0H in excess, 
and boil to expel excess NH 3 , as shown by color change in liq. 
and partial pptn. Add 3-4 mL HOAc (4+1), boil 1-2 min, cool, 
add 10 mL 30% Kl soln, and titr. with std Na 2 S 2 3 soln, 31.040, 
until brown color becomes faint. Add starch indicator, 6.005(f), 
and continue titrn cautiously until blue color due to free I entirely 
disappears. From mL std Na 2 S 2 3 soln used, calc. % Cu in 
sample. 



AOAC Methods (1980) 



Fluorine 



75 



ZINC (ff)-» OFFICIAL FINAL ACTION 

(Applicable to such prepns as Bordeaux-Pb arsenate, Zn arsenite, 

Bordeaux-Zn arsenite, Bordeaux-Paris green, and Bordeaux-Ca 

arsenate) 

6.017 Reagent 

Mercury-thiocyanate soln. — {Caution: See 51.079.) Dissolve 
27 g HgCI 2 and 30 g NH 4 SCN in H 2 and dil. to 1 L 



6.018 



Determination 



Cone, filtrate and washings from sulfide pptn, 6.014, by gentle 
boiling to ca 50 ml_; then evap. on steam bath to dryness. 
Dissolve residue in 100 mL H 2 contg 5 mL HCI, and add 35-40 
mL Hg-thiocyanate soln with vigorous stirring. Let stand ^1 hr 
with occasional stirring. Filter thru weighed gooch, wash with 
H 2 contg 20 mL Hg-thiocyanate soln/L, and dry to const wt at 
105°. Calc. to % Zn, using factor 0.1312. 

Note: Some Fe is usually present and during Zn detn should 
be in ferrous condition. In pptg sulfides pass H 2 S into soln long 
enough to reduce Fe as well as to ppt Cu and Pb. ZnHg(SCN) 4 
ppt normally is white, and occluded Fe(SCN) 3 should not give 
more than faint pink color. 



FLUORINE 
Total Fluorine — Official Final Action 



Lead Chlorofluoride Method (7) 



6.019 



Reagents 

(a) Fusion mixture. — Mix 30 g anhyd. Na 2 C0 3 with 40 g anhyd. 
K 2 C0 3 . 

(b) Lead chlorofluoride wash soln. — Dissolve 10 g Pb(N0 3 ) 2 
in 200 mL H z O, dissolve 1 g NaF in 100 mL H 2 and add 2 mL 
HCI, and mix these 2 solns. Let ppt settle and decant. Wash ppt 
4 or 5 times with 200 mL H 2 by decanting; then add ca 1 L cold 
H 2 to ppt and let stand ^1 hr, with occasional stirring. Filter 
and use clear filtrate. (Prep, more wash soln as needed by 
adding more H 2 to ppt of PbCIF and stirring.) 

(c) Silver nitrate std soln. — 0.1 or 0.2/V. Stdze as in 50.031. 

(d) Potassium or ammonium thiocyanate std soln. — 0.1 N. 
Stdze against std AgN0 3 soln under same conditions as in detn. 

(e) Ferric indicator.— To cold satd Cl-free FeNH 4 (S0 4 ) 2 .12H 2 
soln add enough colorless HN0 3 to bleach brown color. 

(f) Bromophenol blue indicator. — Grind 0.1 g powder with 
1.5 mL 0.1/V NaOH and dil. to 25 mL. 



6.020 



Determination 



(a) Samples difficult to decompose such as cryolite, and 
others that contain aluminum or appreciable amounts of sili- 
ceous material. — Mix 0.5 g sample (or less if necessary to 
contain 0.01-0.10 g F) with 5 g fusion mixt. and 0.2-0.3 g powd. 
Si0 2 in Pt dish, cover with 1 g fusion mixt., and heat to fusion 
over Bunsen burner. (Use of blast lamp is unnecessary since it 
is preferable not to heat much beyond melting temp. If much 
Al is present, uniform, clear, liq. melt cannot be obtained; 
particles of white solid will sep. in melt. Cooled melt should be 
colorless, or at least should not have more than gray color.) 

Leach cooled melt with hot H 2 and when disintegration is 
complete, filter into 400 mL beaker. Return insol. residue to Pt 
dish with jet of H 2 0, add 1 g Na 2 C0 3 , dil. to 30-50 mL, and boil 
few min, disintegrating any lumps with flat-end rod. Filter thru 
same paper, wash thoroly with hot H 2 0, and adjust vol. of filtrate 
and washings to ca 200 mL Add 1 g ZnO dissolved in 20 mL 
HN0 3 (1+9), boil 2 min, stirring constantly, filter, and wash 
thoroly with hot H 2 0. During this washing return gelatinous 



mass to beaker 3 times and thoroly disintegrate in wash soln 
because proper washing of this ppt on filter is difficult. (Mass 
can easily be returned to beaker by rotating funnel above beaker 
while cutting ppt loose from paper with jet of wash soln.) 

Add 2 drops bromophenol blue to filtrate, and with cover 
glass almost entirely over beaker, add HN0 3 (1+4) until color 
just changes to yellow. Make soln slightly alk. with 10% NaOH 
soln, and with cover glass on beaker, boil gently to expel C0 2 . 
Remove from burner; add the HN0 3 until color just changes to 
yellow and then 10% NaOH until color just changes to blue; 
then add 3 mL 10% NaCI soln. (Vol. of soln at this point should 
be ca 250 mL) 

Add 2 mLHCI (1 + 1) and 5 gPb(NO z ) 2 and heat on steam bath. 
As soon as Pb(N0 3 ) 2 is in soln, add 5 g NaOAc, stir vigorously, 
and digest on steam bath 30 min with occasional stirring. Let 
stand overnight, filter, and wash ppt, beaker, and paper once 
with cold H 2 0, then 4 or 5 times with PbCIF wash soln, and then 
once more with cold H 2 0. 

Transfer ppt and paper to beaker in which pptn was made, 
stir paper to pulp, add 100 mL HN0 3 (5+95), and heat on steam 
bath until ppt dissolves. (5 min is ample to dissolve ppt. If 
sample contains appreciable amt of sulfates, ppt will contain 
PbS0 4 , which will not dissolve. In such case heat 5-10 min with 
stirring and consider PbCIF to be dissolved.) Add slight excess 
0.1/V or 0.2/V AgN0 3 , digest on steam bath 30 min, and cool to 
room temp., protecting from light; filter, wash with cold H 2 0, 
and det. AgN0 3 in filtrate by titrn with std thiocyanate soln, 
using 10 mL ferric indicator. Subtract amt of AgN0 3 found in 
filtrate from that originally added. Difference is amt required to 
combine with CI in the PbCIF; from this difference calc. % F in 
sample. 1 mL 0.1/V AgN0 3 = 0.00190 g F. 

(b) Water-soluble fluorides in presence of organic matter. — 
In presence of ^50% org. matter such as flour, pyrethrum, 
tobacco powder, and derris or cube powders, which readily 
decompose without addn of powd Si0 2 and contain little or no 
sulfates, Al, or siliceous compds, mix 0.5 g sample (or less if 
necessary to contain 0.01-0.1 g F) with 5 g fusion mixt., cover 
with 1 g fusion mixt., and heat to fusion over Bunsen burner. 
Leach cooled melt with hot H 2 0, and when disintegration is 
complete, filter into 600 mL beaker. Wash thoroly with hot H 2 
and proceed as in (a), third par. 

In presence of >50% org. matter or org. matter that is 
impractical to free without preliminary ashing, such as apple 
peel and pulp, transfer enough sample to Pt crucible to be 
representative of mixt. and to contain 0.01-0.1 g F. Add 15 mL 
H 2 and enough F-free CaO (0.3-0.4 g) to make mixt. distinctly 
alk. to phthln, mix with glass rod, and evap. to dryness on steam 
bath and in oven at 105°. Ignite at low heat, preferably in furnace 
(^600°), until org. matter is thoroly charred. Pulverize, with glass 
rod, any lumps present in charred ash, mix with 5 g of the fusion 
mixt., and proceed as in (a), first par., beginning ". . . cover with 
1 g fusion mixt., . . /' 

(c) Water-soluble samples in absence of organic matter and 
appreciable quantities of sulfates or aluminum salts. — I n a bsence 
of org. matter or other interfering substances, fusion may be 
omitted and detn made on aliquot of aq. soln contg 0.01-0.1 g 
F, as in (a), third par. 

In presence of Al, as in samples contg Na 2 SiF 6 and 
KAI(S0 4 ) 2 .12H 2 0, transfer sample to 400 mL beaker, dissolve in 
150 mL hot H 2 0, add 6 g fusion mixt., and boil. Add 1 g ZnO 
dissolved in 20 mL HN0 3 (1+9), boil 2 min with const stirring, 
filter into 500 mL vol. flask, and wash thoroly with hot H 2 0. Cool 
to room temp, and dil. to vol. Transfer 200 mL aliquot contg 
0.01-0.10 g F to 600 mL beaker and proceed as in (a), third par. 

(d) Sodium and magnesium fluosilicates, or samples con- 
taining more than 5% sulfates in absence of aluminum and 



76 



6. Pesticide Formulations 



AOAC Methods (1980) 



boron, with or without moderate amounts of organic matter. — 
With large amts of Na 2 StF 6 and some other more volatile 
fluosilicates, e.g., MgSiF 6 , where there is possibility of some F 
being evolved as SiF 4 before fusion is effected, or in samples 
contg appreciable amts of sulfates, distil F as in 6.024, and det. 
F in distillate as follows: Add several drops bromophenol blue, 
make alk. with NaOH, and adjust vol. to ca 250 mL by gently 
boiling down vol. from 400 to 250 mL. Proceed as in (a), third 
par., beginning "Remove from burner; . . ." 

Notes: These methods give accurate results for 0.01-0.10 g 
F. Below 0.01 g, results tend to be slightly low, and above 0.10 
g, slightly high. Convenient sample to fuse is one contg 0.07-0.08 
g F; too large sample may result in incomplete fusion. Large 
amts of B compds and alkali salts retard or prevent complete 
pptn of PbCIF. B has greater effect when amt of F is large than 
when it is small. In methods described B has little effect and it 
may be disregarded in analysis of insecticides if amt of F to be 
pptd is ^0.03 g. With some prepns contg Na 2 B 4 7 or H 3 B0 3 , 
where it is difficultto obtain representative mixt. when extremely 
small sample (0.1 g) is used for analysis, take larger sample and 
ppt PbCIF from aliquot of fusion sola Amt of alkali carbonates 
specified in fusion and in washing of insol. residue is not large 
enough to cause low results. If sample contains S, remove it 
with CS 2 and det. F on air-dried residue, allowing in calcns for 
% S removed. {Caution: See 51.039, 51.040, and 51.048.) 

Modified Travers Method (S) 

(Applicable in absence of B, Al, and 
large amts of pyrethrum powder) 



6.021 



Reagents 



(a) Alcohoiic potassium chloride so/n.— Dissolve 60 g KCI in 
400 mL H 2 0, add 400 mL alcohol, and test with phthln; if soln 
is not neut, adjust to exact neutrality with NaOH or HCI soln. 

(b) Sodium hydroxide std soin. — 0.2/V. Prep, and stdze as in 
50.032—50.035. 



6.022 



Determination 



Treat 0.5 g sample in small beaker with 20-25 mL H 2 0. Add 
0.3 g finely divided pptd Si0 2 and few drops Me orange. Add 
HCI dropwise until soln assumes apparently permanent pink; 



then add 2 mL excess, cover beaker with watch glass, and boil 
1 min. Cool to room temp., add 4 g KCI, and stir until KCI 
dissolves. Add 25 mL alcohol and let stand 1 hr, stirring 
frequently. Filter thru gooch contg disk of filter paper covered 
with medium pad of asbestos. Wash ppt with ale. KCI soln until 
one washing does not destroy color made by 1 drop 0.2/V NaOH 
and phthln (usually 3-4 washings). Transfer crucible and con- 
tents to 400 mL.beaker, add 100 mL recently boiled H 2 and 1-2 
mL phthln, heat, and titr. with std NaOH soln. Finish titrn with 
the F soln actively boiling. Calc. % F. 1 mL 0.2/V NaOH - 0.0057 
QF. 

Distillation Method [9) 

(Applicable to H 2 0-sol. or H 2 0-insol. insecticides 
in absence of gelatinous Si0 2 , B, and Al) 

6.023 Reagents 

(a) Sodium alizarin sulfonate indica tor. —Dissolve 0.1 g Na 
alizarin sulfonate in 200 mL H 2 0. 

(b) Thorium nitrate soln. — Approx. 0.05/V. Stdze in terms of 
g F/mL by titrg F obtained by distn from std NaF as in 6.024. In 
stdzg for use with 6.024(b), add 5 mL satd KMn0 4 soln in addn 
to other reagents in distn flask. 



6.024 



Determination 



{a) In absence of organic matter. — Weigh sample contg ca 
0.09 g F, and with aid of little H 2 transfer to 250 mL Claisen 
distn flask contg 12 glass beads. Adjust to ca 30 mL and close 
flask with 2-hole rubber stopper, thru which pass thermometer 
and 4 mm glass tube, both of which extend into soln. (The 4 
mm glass tube extends ca 5 cm above rubber stopper and by 
means of rubber tube, £, connects still with 1 L Florence flask 
contg H 2 for steam generation. Flask is equipped with steam 
discharge, H, and pressure tube, G. See Fig. 6:03.) 

Bring H 2 in steam generating flask to boil with pinchcock, F , 
in release tube open. Connect distg flask to condenser, and add 
25 mL H 2 S0 4 thru top of 4 mm tube, using pipet or special 
funnel. With pinchcock, F, open, connect rubber tubing to 4 mm 




FIG. 6:03— Apparatus for determining fluorine 



AOAC Methods (1980) 



Herbicide Volatility 



77 



tube. Light burner under Claisen flask. Regulate flow of steam 
by adjusting burner flames and pinchcock, F, so that vol. of soln 
is held const and temp, in flask, B, is kept at 145-150°. 
Continue distn until 400 mL distillate collects. DM. to 500 ml_ in 
vol. flask, transfer 50 mL aliquot to tall-form 1 50 mL beaker, and 
add 5 drops indicator, 6.023(a). Adjust acidity with 1% NaOH 
soln and HCI (1 +249) until pink just disappears. Add 2 mL of the 
HCI, and titr. with 0.05/V Th(N0 3 ) 4 to permanent pink, using buret 
graduated in 0.05 mL. 

(b) In presence of organic matter. — {Caution: See 51.080.) In 
presence of moderate amts of org. matter, transfer sample contg 
ca 0.09 g F and contg =s0.2 g org. matter, with aid of little H 2 0, 
to 250 mL Claisen distn flask contg 12 glass beads. Add 5 mL 
satd KMn0 4 soln, adjust to ca 30 mL, and proceed as in (a), 
beginning ". . . close flask with 2-hole rubber stopper, . . ." 

In presence of large amts of org. matter, transfer sample to 
medium-size Pt dish, add 15 mL H 2 and enough F-free CaO to 
make mixt. distinctly alk. to phthln, mix with glass rod, and 
evap. to dryness on steam bath and in oven at 105°. Ignite at 
low heat, preferably in furnace (^600°), until org. matter is 
thoroly charred. Pulverize any lumps present in charred ash with 
glass rod, transfer to 250 mL Claisen distn flask by brushing, 
and finally wash out dish with 30 mL H 2 S0 4 {1 +9). Except to add 
22 mL instead of 25 mL H 2 S0 4 , proceed as in (a), par. 2. 

Note: If coating of pptd Si0 2 forms on inside of distn flask, 
remove by treatment with hot coned alkali soln, as it is capable 
of retaining F during distn of some samples and giving it up, at 
least in part, in later distns. 



Fluorine Present as Sodium Fluosilicate 
Official Final Action 



(B, CaO, and alum absent) 



6.025 



Reagents 

Alcoholic potassium chloride and sodium carbonate soln.— 
Dissolve 1.0 g Na 2 C0 3 in 100 mL ale. KCI reagent, 6.021(a). 
For other reagent see 6.021. 



(b) Mobile solvents. — (7) n-Hexane, (2) fl-hexane-acetone . 
(98+2), and (3) /?-hexane-alcohol (98 + 2). 

(c) Pesticide std solns. — 1 ng/fiL EtOAc, acetone, or any 
convenient solv. 

6.029 Preparation of Sample 

Ext 8 g sample with 20 mL acetone in 250 mL erlenmeyer by 
shaking intermittently 5 min. Let solids settle. If soln is turbid, 
filter or centrf. to obtain clear supernate for spotting. For samples 
contg large amts S, use 8 g sample and 20 mL pet ether. 

6.030 Preparation of Plates 

Dissolve 0.1-0.15 g AgN0 3 in 1-2 mL H 2 in 100 mL beaker, 
add 58 mL MeOH, and mix. Weigh 40 g adsorbent, (a), in 250 
mL flask, add AgN0 3 -MeOH soln, and shake vigorously 20 sec. 
Apply slurry as 0.25 mm thick layer to five 20 x 20 cm (8 x 8") 
plates positioned on plastic mounting board. After plates appear 
dry, store in desiccator overdesiccant. When plate is dry, scrape 
1 cm strip from side edges to ensure even solv. front. Use plate 
immediately after removal from desiccator. 



6.031 



Detection 



Pour n-hexane into glass chromatgc tank to depth of 10-20 
mm. Place 2 paper blotters (ca 7.5 x 22 cm) on each side of tank 
or large blotter covering back of tank and let equilibrate ^2 hr 
before use. 

Spot 10 jul sample ext on plate with 100 /aL syringe. Do not 
disturb adsorbent layer. Also spot std solns of pesticides de- 
clared as part of formulation. Spots should be ^6 mm diam. 
and placed <30 mm from bottom of plate. Place plate in 
chromatgc tank, and let plate develop 2*10 cm. Remove plate 
and expose to shortwave UV, 6.027(b). {Caution: See 51.016.) 
Chlorinated org. pesticides should be visible as dark spots 
against white or light gray background. Expose plates s^1 hr. 
Longer exposure will not harm plates. 

To confirm identification of pesticide, repeat TLC step with 
different mobile solv., 6.028(b)(2) or (5). 



6.026 



Determination 



Weigh 1 g sample into Pt dish, and add rapidly, with continuous 
stirring, 50 mL of the ale. KCI-Na 2 C0 3 reagent. Do not let soln 
become acid, and if necessary, use more reagent to insure alky. 
Continue stirring until all sol. portions of sample dissolve. 
Proceed as in 6.022, beginning: "Filter thru gooch . . ." Calc. 
% Na 2 SiF 6 (1 mL 0.2/V NaOH - 0.009403 g Na 2 SiF 6 ). 



CONTAMINATION BY ORGANOCHLORINE 
PESTICIDES {/0)-OFFIC6AL FINAL ACTION 

AOAC-CIPAC Method 

(Applicable to detection of contamination by 0.01% chlorinated 

hydrocarbons such as aldrin, DDT, dieldrin, and endrin, and 

0.05-0.10% of chlordane, Strobane, and toxaphene) 

6.027 Apparatus 

(a) Thin layer chromatographic apparatus.— See 29.006. 

(b) Ultraviolet apparatus.— §\er\\arr\p G-15T8 (Westinghouse 
Electric Corp., Lamp Divisions, One Westinghouse Plaza, Bloom- 
field, NJ 07003). 

6.028 Reagents 

(a) Adsorbent. — Aluminum oxide G, Type E (Brinkmann In- 
struments, Inc.). 



VOLATILITY OF ESTER FORMS OF HORMONE-TYPE 
HERBICIDES {//)— OFFICIAL FINAL ACTION 

6.032 Material 

(a) Paper bags. — No. 20 to open with flat bottom. Close with 
paper clips. 

(b) Filter paper. — 7 cm diam. 

(c) Bacteriological loop. — 0.01 mL. Wash with acetone after 
each application or heat to cherry red in flame. 

(d) Test plants. — Actively growing tomato seedlings 65-75 
mm high in 3-4" pots. 

(e) Formulation to be tested. — Use 0.01 mL aliquot of 4 lb/gal. 
formulation or equiv. vol. of other conens. 

(f) High and low volatile ester stds. — Use Bu ester of 2,4-D as 
high volatile ester and tetrahydrofurfural ester of 2,4-D as low 
volatile ester with same wt of acid/gal. as formulations to be 
tested. 

6.033 Operating Technic 

Open bags with flat bottom and place plant toward one side 
on bottom of bag. Apply 0.01 mL of formulation to middle of 
filter paper by means of bacteriological loop, and for controls, 
apply 0.01 mL solv. only. Place treated paper in bottom of bag. 
Do not touch treated part of paper against plant, sides of bag, 
or pot. Close bag by folding top, secure with clips, and let stand 
24hrat85-110°F(29-43°C). 



78 



6. Pesticide Formulations 



AOAC Methods (1980) 



Use 3 plants per treatment and 3 for controls. Repeat test on 
another day. 

Remove plants from bag, let stand 24 hr, and read curvature 
(stem bending, epinasty) response. (Fold and discard used bags 
to prevent contamination.) Rate plants according to scale as 
follows: 

(1 ) Normal growth of untreated check-no apparent response. 

(2) Epinasty 1-20° compared to normal-no curling. 

(3) Epinasty 21-40° compared to normal-slight curling. 

(4) Epinasty 41-60° compared to normal-moderate curling. 

(5) Epinasty 61-80° compared to normal-moderate curling. 

(6) Epinasty 81 to >90° compared to normal-severe formative 
effects. 

Mean response of 1 to 2.4 for all tests indicates low volatility. 
Mean response of 2.5 to 6 indicates volatile formulation. 

To detect small differences between low volatile esters, or 
differences between 2,4-D and 2,4,5-T types, hold plants 7 days 
after treatment to allow time for modified leaves or stem lesions 
to develop. Absence of such responses indicates that formulation 
was a low volatile 2,4,5-T ester. 



INORGANIC AND ORGANOMETALUC 
PESTICIDES AND ADJUVANTS 

• PARIS GREEN • 

{Caution: See 51.041.) 

6.034 Moisture— Official Final Action 
See 6.004. 

6.035 Total Arsenic — Official Final Action 
See 6.007. 

Total Arsenious Oxide — Official Final Action 

(Following methods det. only As present in trivalent form (As 2 3 ) 

and Sb present in trivalent form (Sb 2 3 ) in absence of ferrous 

and cuprous salts.) 



Method I {12) 
6.036 
See 4.028, 10th ed. 

6.037 

lodometric titrn. See 4.029, 10th ed. 

6.038 Method II {13) 

Bromate titrn. See 4.030, 10th ed. 

6.039 Water-Soluble Arsenious Oxide 
Official Final Action 

lodometric titrn. See 4.031, 10th ed. 



Total Copper — Official Final Action 
6.040 Electrolytic Method 

See 4.032, 10th ed. 



Reagents 



Determination 



LEAD ARSENATE 

{Caution: See 51.041 and 51.078.) 

6.042 Moisture — Official Final Action 

(a) Powder.— Dry 2 g to const wt at 105-110°. Report loss in 
wt as H 2 0. 

(b) Paste. — Proceed as in (a), using 50 g. Grind dry sample to 
fine powder, mix well, transfer small portion to sample bottle, 
and again dry 1-2 hr at 105-110°. Use this anhyd. material to 
det. total Pb and total As. 

Total Arsenic — Official Final Action 

6.043 Method I 
See 6.007 

6.044 Method II {15) 

(Not applicable in presence of Sb) 
Dissolve 1 g powd sample with HN0 3 (1+4) in porcelain 
casserole or evapg dish, add 5 mL H 2 S0 4 , and heat on hot plate 
to copious evolution of white fumes. Cool, add little H 2 0, and 
again evap. until white fumes appear, to assure removal of last 
trace of HN0 3 . Wash into 200 mL vol. flask with H 2 0, cool, dil. 
to vol., and filter thru dry filter. Transfer 100 mL filtrate to 
erlenmeyer and proceed as in 6.013, beginning ". . . add 1 g Kl, 
. . ." From mL std I soln used, calc. % total As as As 2 5 . 

6.045 Total Arsenious Oxide {76) — Official Final Action 

Weigh 2 g powd sample and transfer to 200 mL vol. flask, add 
100 mL H 2 S0 4 (1+6), and boil 30 min. Cool, dil. to vol., shake 
thoroly, and filter thru dry filter. Nearly neutze 100 mL filtrate 
with NaOH soln, 6.005(e), using few drops phthin. If neut. point 
is passed, make acid again with the dil. H 2 S0 4 . Continue as in 
6.013, beginning "Neutze with NaHC0 3 , . . ■" From mL std I soln 
used, calc. % As 2 3 . 

Total Arsenic Oxide {17) — Official Final Action 

6.046 Reagents 

(a) Potassium iodide soln. — Dissolve 20 g Kl in H 2 and dil. 
to 100 mL 

(b) Thiosulfate std soln.-0.0SN. Prep, daily by dilg 0.1/V 
soln, 50.037-50.038. 1 mL 0.05/V Na 2 S 2 3 = 2.873 mg As 2 5 - 



6.047 



Determination 



Weigh 0.5 g powd sample and transfer to erlenmeyer. Add 
25-30 mL HCl and evap. to dryness on steam bath. Add 50 mL 
HCI and if necessary to effect soln, heat on steam bath, keeping 
flask covered with watch glass to prevent evapn of acid. Cool 
to 20-25°, add 10 mL of the Kl soln and 50 mL (or more if 
necessary to produce clear soln) 25% NH£i soln f and imme- 
diately titr. liberated i with std Na 2 S 2 3 soln. When color becomes 
faint yellow, dil. with ca 150 mL H 2 and continue titrn carefully, 
dropwise, until colorless, using starch indicator, 6.005(f), near 
end point. From mL Na 2 S 2 3 soln used, calc. % As 2 5 . 

6.048 Water-Soluble Arsenic — Official Final Action 

Proceed as in 6.013, and calc. results as As 2 5 . 



6.041 Volumetric Thiosulfate Method [14) 
See 4.033, 10th ed. 

it Methods for this product are surplus — see inside front cover. 



6.049 Total Lead {18)— Official Final Action 

In 600 mL beaker on hot plate heat 0.5 g powd sample and ca 
25 mL HN0 3 (1+4). Filter to remove any insol. residue. Dil. to 
5=400 mL, heat nearly to bp, and add NH 4 OH to slight pptn, then 



AOAC Methods (1980) 



Copper Naphthenate 



79 



HN0 3 (1+9) to redissolve ppt, adding 1-2 ml_ excess. Into this 
soln, kept almost boiling, pipet 50 ml_ hot 10% K 2 CrO A soln, 
stirring constantly. Decant while hot thru weighed gooch, pre- 
viously heated to 140-150°, and wash ppt several times by 
decanting and then on filter with boiling H 2 until washings are 
colorless. Dry PbCr0 4 at 140-150° to const wt. From wt PbCr0 4 , 
calc. % Pb, using factor 0.6411. (PbCr0 4 ppt may contain small 
amt PbHAs0 4 , which will cause slightly high results, but this 
error rarely is >0.1-0.2%.) 



let stand 3 hr on steam bath. Filter, and wash with hot H 2 0. 
Ignite at 950°, and weigh as CaO; or dissolve and titr. as in 
6.055. From wt CaO or ml_ KMn0 4 soln used, calc. % Ca. 

* ZINC ARSENITE * 
(Caution: See 51.041.) 

6.057 Moisture — Official Final Action 
See 6.004. 



CALCIUM ARSENATE 

(Caution: See 51.041.) 

6.050 Moisture — Official Final Action 
See 6.004. 

6.051 Total Arsenic — Official Final Action 
See 6.007. 

6.052 Total Arsenious Oxide [19) — Official Final Action 

( a ) Not appiicabie in presence of nitrates. — We ighlgsample, 
transfer to 500 mL erlenmeyer, and dissolve in 100 mL HCI 
(1+3). Heat to 90° and titr. with std KBr0 3 soln, 6.005(c), using 
10 drops Me orange, 6.005(g). From mL std KBr0 3 soln used, 
calc. % As 2 3 . 

(b) Appiicabie in presence of smaii amounts of nitrates. — 
Proceed as in (a) except to titr. at room temp. 

6.053 Water-Soluble Arsenic — Official Final Action 

Proceed as in 6.013, and calc. results as As 2 5 . (In testing Ca 
arsenate by this method, low value for H 2 0-sol. As is not 
assurance against plant injury when using this product.) 



Total Calcium (79) — Official Final Action 



6.054 



Reagents 

(a) Ammonium oxaiate soin. — Dissolve 40 g (NH 4 ) 2 C 2 4 .H 2 
in 1 L H 2 0. 

(b) Potassium permanganate std soin. — 0.1 N. Prep, and 
stdze as in 50,025-50.026. 

6.055 Method I 

Dissolve 2 g sample in 80 mL HOAc (1+3), transfer to 200 mL 
vol. flask, dil. to vol., and filter thru dry filter. Transfer 50 mL 
aliquot to beaker, dil. to ca 200 mL, heat to bp, and ppt Ca with 
(NH 4 ) 2 C 2 4 soln. Let beaker stand 3 hr on steam bath, filter, and 
wash ppt with hot H 2 0. Dissolve ppt in 200 mL H 2 contg 25 mL 
H 2 S0 4 (1+4), heat to ca 70°, and titr. with std KMn0 4 soln. From 
mL KMn0 4 soln used, calc. % Ca. 

6.056 Method II 

(Not applicable in presence of Pb. Caution: See 51.078.) 

Weigh 2 g sample, transfer to beaker, add 5 mL HBr (ca 1.38 
sp gr) and 15 mL HCI, and evap. to dryness under hood to 
remove As. Repeat treatment, add 20 mL HCI, and again evap. 
to dryness. Take up with H 2 and little HCI, filter into 200 mL 
vol. flask, wash, and dil. to vol. Transfer 50 mL aliquot to beaker, 
add 10 mL HCI and few drops HN0 3 , boil, and make slightly alk. 
with NH 4 OH. Let stand few min and filter. Dissolve ppt in HCI 
(1+4), reppt, filter thru same paper, and wash with hot H 2 0. To 
combined filtrates and washings add 20 mL HOAc (1+3) and 
adjust to ca 200 mL. Heat to bp, ppt with (NH 4 ) 2 C 2 4 soln, and 



6.058 Total Arsenic — Official Final Action 

Proceed as in 6.007 and calc. as As 2 3 . 

Total Arsenious Oxide— Official Final Action 

6.059 Method I [19] 

Bromate titrn. See 4.051, 10th ed. 

6.060 Method II 

lodometric titrn. See 4.052, 10th ed. 

6.061 Water-Soluble Arsenic — Official Final Action 

Proceed as in 6.013, and calc. results as As 2 3 . 

6.062 Total Zinc (7S)~ Official Final Action 

Gravimetric method. See 4.054, 10th ed. 

it COPPER CARBONATE i*r 
Copper — Official Final Action 

6.063 Electrolytic Method 
See 4.055, 10th ed. 

6.064 Volumetric Thiosulfate Method 
See 4.056, 10th ed. 

COPPER NAPHTHENATE 

(Caution: See 51.041.) 

Copper (20) — Official First Action 

6.065 Titrimetric Method 

Accurately weigh sample contg ca 0.2 g Cu into dry g-s flask. 
Add 5 mL pet ether to coned products. Add 100 mL H 2 0, 1.5 g 
NH A HF 2 , and 5-10 g Kl. Stopper and shake vigorously until 
reaction is complete (usually ca 2 min). Wash stopper and sides 
of flask with H 2 and titr. with std 0.1/V Na 2 S 2 3 (stdzd against 
Cu) to light brown. Add starch indicator, 6.005(f), titr. almost to 
end point, add 2 g KSCN, shake to dissolve, and complete titrn 
to starch end point. 

6.066 Electrolytic Method 

Accurately weigh sample contg ca 0.2 g Cu into 200 mL 
separator. Add 50 mL pet ether and 25 mL HN0 3 (1+4), and 
shake 2 min. Drain aq. phase into 250 mL beaker and save. Wash 
pet ether with 15 and 10 mL HN0 3 (1+4), and combine acid 
exts. Neutze with NH 4 OH, acidify with 6 mL H 2 S0 4 and 4 mL 
HNO3, and proceed as in 6.015, beginning ". . . adjust vol. to 200 
mL, . . ." using ca 0.5 amp during first 10 min and 1.5-2.0 amp 
for ca 20 min. 

it Methods for this product are surplus— ^ee inside front cover. 



80 



6. Pesticide Formulations 



AOAC Methods (1980) 



BORDEAUX MIXTURE 

{Caution: See 51.041.) 

6.067 Moisture— Official Final Action 

(a) Powder.— See 6.042(a). 

(b) Paste. — Heat ca 100 g in oven at 90-100° until dry enough 
to powder readily and note loss in wt. Powder this partially dried 
sample and det. remaining H 2 in 2 g as in (a). Det. C0 2 as in 
6.069, both in original paste and in partially dried sample. Calc. 
total H 2 by following formula: 

M _ n , (100 -a)fr+c) . 

where M = % total H 2 in original paste; a = % loss in wt of 
original paste during first drying; b = % loss in wt of partially 
dried paste during second drying; c - % C0 2 remaining in 
partially dried paste after first drying; and d = % total C0 2 in 
original paste. 



Carbon Dioxide (27)— Official Final Action 



6.068 



Apparatus 



Use 200 mL erlenmeyer with 2-hole stopper; in one hole fit 
dropping funnel with stem extending almost to bottom of flask, 
and thru other hole pass outlet of condenser that is inclined 
upward at 30° angle from horizontal. Connect upper end of 
condenser with CaCI 2 tube, which in turn connects with double 
U-tube filled in middle with pumice fragments, previously satd 
with 20% CuS0 4 .5H 2 soln and subsequently dehydrated, and 
with CaCI 2 at either end. Connect 2 weighed U-tubes to absorb 
C0 2 , first filled with porous soda-lime, and second, !/ 3 with soda- 
lime and 2 / 3 with CaCI 2 , placing the CaCI 2 at exit end of train. 
Attach Geissler bulb, partly filled with H 2 S0 4 , to last U-tube to 
show rate of gas flow, and connect aspirator with Geissler bulb 
to draw air thru app. Connect absorption tower filled with soda- 
lime to mouth of dropping funnel to remove C0 2 from air 
entering app. 



6.069 



Determination 



Weigh 2 g powder or 10 g paste into the erlenmeyer and add 
ca 20 mL H 2 0. Attach flask to app., omitting the 2 weighed U- 
tubes, and draw C0 2 -free air thru app. until it displaces original 
air. Attach weighed U-tubes as in 6.068, close stopcock of 
dropping funnel, pour into it 50 mL HCI (1+4), reconnect with 
soda-lime tower, and let acid flow into erlenmeyer, slowly if 
there is much C0 2 , rapidly if there is little. When effervescence 
diminishes, place low Bunsen flame under flask and start flow 
of H 2 thru condenser, letting slow current of air flow thru app. 
at same time. Maintain steady but quiet boil and slow air current 
thru app. Boil few min after H 2 begins to condense, remove 
flame, and continue air flow at ca 2 bubbles/ sec until app. is 
cool. Disconnect weighed absorption tubes, cool in balance 
case, and weigh. Increase in wt = C0 2 . 

Copper — Official Final Action 

6.070 Electrolytic Method 

(Also applicable to CuC0 3 and CuS0 4 ) 
Dissolve powd sample contg 0.2^0.25 g Cu in 45 mL HN0 3 
(1 +4). Filter if necessary, dil. to 200 mL, and electrolyze as in 
6.015. 

6.071 Volumetric Thiosulfate Method 

Dissolve 2 g powd sample in ca 25 mL HN0 3 (1+4), dil. to 50 
mL, add NH 4 OH in excess, and heat. Without removing ppt that 



has formed, boil off excess NH 3 , add 3-4 mL HOAc, cool, add 
10 mL 30% Kl soln, and titr. as in 6.016, beginning ". . . titr. with 
std Na 2 S 2 3 soln, . . ." 



* BORDEAUX MIXTURE WITH PARIS GREEN * 

{Caution: See 51.041.) 

6.072 Moisture — Official Final Action 
See 6.067. 

6.073 Carbon Dioxide — Official Final Action 
See 6.069. 

6.074 Total Arsenic — Official Final Action 

Proceed as in 6.007, using 2 g sample, and calc. results as 
As 2 3 . 

6.075 Total Arsenious Oxide— Official Final Action 

lodometric titrn. See 4.067, 10th ed. 

6.076 Water-Soluble Arsenious Oxide — Official Final Action 

lodometric titrn. See 4.068, 10th ed. 

Copper — Official Final Action 

6.077 Electrolytic Method I 
See 6.015. 

6.078 Electrolytic Method II— [Short Method) 
See 4.070, 10th ed. 

6.079 Volumetric Thiosulfate Method 
See 6.016. 



* BORDEAUX MIXTURE WITH LEAD ARSENATE * 

{Caution: See 51.041.) 

6.080 Moisture — Official Final Action 
See 6.067. 

6.081 Carbon Dioxide — Official Final Action 
See 6.069. 

6.082 Total Arsenic— Official Final Action 

Proceed as in 6.007, using 2 g sample, and calc. results as 
As 2 5 . 

6.083 Water-Soluble Arsenic — Official Final Action 

Proceed as in 6.013 and calc. results as As 2 5 . 

Copper — Official Final Action 

6.084 Electrolytic Method 
See 6.015. 



* Methods for this product are surplus — see inside front cover. 



AOAC Methods (1980) 



Lime Sulfur 



81 



6.085 Volumetric Thiosulfate Method 
See 6.016, 

6.086 Lead — Official Final Action 
See 6.014. 

Lead and Copper — Official Final Action 

6.087 Electrolytic Method (22) 

(Caution: See 51.026, 51.047, and 51.078.) 
See 4.07M.080, 10th ed. 

* BORDEAUX MIXTURE WITH CALCIUM ARSENATE * 

{Caution: See 51.041.) 

6.088 Moisture — Official Final Action 
See 6.067. 

6.089 Carbon Dioxide— Official Final Action 
See 6.069. 

6.090 Total Arsenic — Official Final Action 

Proceed as in 6.007, using 2 g sample, and calc. results as 
As 2 5 . 

6.091 Water-Soluble Arsenic — Official Final Action 

Proceed as in 6.013 and calc. results as As 2 5 . 

Copper — Official Final Action 

6.092 Electrolytic Method I 
See 6.015. 

6.093 Electrolytic Method II 
See 6.078. 

6.094 Volumetric Thiosulfate Method 
See 6.016. 

* CALCIUM CYANIDE (23) • 

6.095 Cyanide— Official Final Action 

AgN0 3 titrn. See 4.093-4.094, 10th ed. 

Chloride — Official Final Action 

6.096 Method I 
See 4.095, 10th ed. 

6.097 Method II 
See 4.096, 10th ed. 

POTASSIUM CYANATE (24)— OFFICIAL FINAL ACTION 

{Caution: See 51.041.) 

6.098 Reagent 

Wash soln. — Satd aq. soln of hydrazodicarbamide, NH 2 CO- 
NHNHCONH 2 . Prep, by mixing KOCN and semicarbazide.HCI, 
NH 2 CONHNH 2 .HCI, in H 2 0, filter, and wash ppt with H 2 0. Transfer 
ppt to flask, add small amt H 2 0, shake vigorously, and filter. 
(Solubility of ppt in H 2 is ca 1 part in 6600.) 



6.099 



Determination 



Weigh sample contg 0.2-0.5 g KOCN into 100 mL beaker, add 
20 mL wash soln and 1 g semicarbazide.HCI, and let stand 24 
hr. Filter hydrazodicarbamide on gooch or fine fritted glass 
crucible, wash with 10 mL wash soln, and dry at 100° to const 
wt. KOCN = wt residue x 0.6868. 

* SODIUM AND POTASSIUM CYANIDES * 

(Caution: See 51.050.) 

6.100 Cyanide (25)— Official Final Action 

AgN0 3 titrn. See 4.088-4.089, 10th ed. (Caution: See 51.084.) 

Chloride (26)— Official Final Action 

6.101 Method I 

Pptn with AgN0 3 and thiocyanate back-titrn. See 4.090-4.091, 
10th ed. 

6.102 Method II 

Distn, pptn with AgN0 3 , and thiocyanate back-titrn. See 4.092, 
10th ed. 

LIME SULFUR SOLUTIONS AND DRY LIME SULFUR 

Soluble Sulfur (27)— Official Final Action 

(Use low S reagents.) 

6.103 Preparation of Sample 

(a) Soins. — Accurately weigh ca 10 g soln, transfer to 250 mL 
vol. flask, and immediately dil. to vol. with recently boiled and 
cooled H 2 0. Mix thoroly and either take necessary aliquots in 
individual pipets in min. time for detns or transfer to small 
bottles, filling them completely and avoiding contact of soln 
with air as much as possible. Stopper bottles, seal with paraffin, 
and store in dark, cool place. 

(b) Dry lime-sulfur. — Thoroly stir 5 g sample with ca 50 mL 
H 2 in 250 mL beaker. Let settle and decant thru paper into 250 
mL vol. flask. Repeat extn with H 2 until filtrate is colorless and 
ca 200 mL is obtained. Transfer residue to paper, wash with hot 
H 2 0, cool to room temp., and dil. to vol. Dry residue 1.5 hr at 
105°, and reserve for free S and sulfite S detns in residue, if 
desired. (Ext S from dry residue with CS 2 (Caution: See 51.039, 
51.040, and 51.048), evap. on steam bath or in air current, dry 
15 min at 105°, weigh, and calc. % S.) 

Prep, soln in min. time and keep beaker and funnel covered 
as much as possible. 



6.104 



Determination 



With clean, dry pipet transfer 10 mL prepd soln, 6.103(a) or 
(b), to 250 mL beaker. Partially cover with cover glass and add 
2-3 g Na 2 2 in small portions, with stirring, from tip of spatula. 
Continue adding Na 2 2 until all S appears to be oxidized to 
sulfate (yellow color disappears). Add slight excess Na 2 2 , 
completely cover beaker with cover glass, and heat on steam 
bath, stirring occasionally, 15-20 min. 

Wash off cover glass and sides of beaker, acidify with HCI 
(1 +4), and filter if necessary. Dil. to 1 50-200 mL, heat to bp, and 
add 10% BaCksoln (11 mL/1 g BaSOJ, with const stirring, at 
such rate that ca 4 min is required to add necessary amt. Let 
stand until clear and cool, filter thru quant, paper, wash until Cl- 
free, ignite carefully, and heat to const wt over Bunsen burner. 
Calc. % S from wt BaS0 4 , using factor 0.1374. 

* Methods for this product are surplus— see inside front cover. 



82 



6. Pesticide Formulations 



AOAC Methods (1980) 



Thiosulfate Sulfur (27)— Official Final Action 

6.105 Reagent 

Ammoniacal zinc chloride soln. — Dissolve 50 g ZnCI 2 in ca 500 
mL H 2 0, add 125 mL NH 4 OH and 50 g NH 4 CI, and dil. to 1 L 



6.112 



Deter mina t/'on 



6.106 



Determination 



To 50 mL H 2 in 200 mL vol. flask add 50 mL prepd soln, 
6.103(a) or (b). Add slight excess of the ammoniacal ZnCI 2 soln 
and dil. to vol. Complete detn as rapidly as possible. Shake 
thoroly and filter thru dry fitter. To 100 mL filtrate add few drops 
Me orange, 6.005(g), or Me red, 2.055(i), and exactly neutze with 
0.1/V HCI. Titr. neut. soln with 0.05/V I, 6.005(b), using few drops 
starch indicator, 6.005(f). From mL I soln used, calc. % thiosulfate 
S present. (Factor of I soln in terms of As 2 3 x 1.296 = equiv. 
in thiosulfate S.) 



Sulfide Sulfur— Official Final Action 

6.107 Zinc Chloride Method [27) 

To 10-15 mL H 2 in small beaker add 10 mL aliquot prepd 
soln, 6.103(a) or (b). Calc. amt ammoniacal ZnCI 2 sotn, 6.105, 
necessary to ppt ail S in aliquot and add slight excess. Stir 
thoroly, filter, wash ppt twice with cold H 2 0, and transfer paper 
and ppt to beaker in which pptn was made. Cover with H 2 0, 
disintegrate paper with glass rod, and add ca 3 g Na 2 O z , keeping 
beaker well covered with watch glass. Warm on steam bath with 
frequent shaking until all S is oxidized to sulfate, adding more 
Na 2 2 if necessary. Acidify slightly with HCI (1+4), filter to 
remove shreds of paper, wash thoroly with hot H 2 0, and det. S 
in filtrate as in 6.104. 

6.108 Indirect Method 

Difference between sol. S and sum of thiosulfate S and sulfate 
S = sulfide S. 



Transfer 20 mL sample to 1 L vol. flask and dil. to vol. Pipet 
50 mL aliquot of mixt. into 200 mL erlenmeyer. Add excess 
As 2 3 soln and then decided excess NaHC0 3 . Titr. excess As 2 3 
with std I soln, using starch soln, 6.005(f), or the I as its own 
indicator. Subtract vol. I soln, corrected to 0.1 N, from vol. As 2 3 
soln used, and from this value and sp gr of soln, calc. % NaOCI. 
1 mL 0.1/V As 2 3 = 0.003722 g NaOCI. 

6.113 Available Chlorine — Official Final Action 

Calc. % available CI from titrn, 6.112. 1 mL 0.1 N As 2 3 = 
0.003545 g available CI. 

6.114 Chloride Chlorine — Official Final Action 

Pipet 50 mL aliquot prepd soln, 6.112, into 200 mL erlenmeyer 
and add slight excess As 2 3 soln, 6.111(a), calcd from NaOCI 
titrn; add slight excess HN0 3 , neutze with CaC0 3t and titr. with 
0.1/V AgN0 3 , 50.027-50.029, using K 2 Cr0 4 sola 50.028(b), or the 
Ag 3 As0 4 formed in soln, as indicator. Det. blank on reagents 
and correct for any CI found. From this corrected titrn and sp gr 
of sample, calc. % CI. From this value subtract 1 / 2 the % available 
CI. Difference = % chloride CI. 

6.115 Sodium Hydroxide [29)— Official Final Action 

Stdze pH meter equipped with calomel and glass electrodes, 
using std pH 6.9 buffer soln, 50.007(d). 

Place 50 mL 10% BaCI 2 .2H 2 soln and 30 mL 3% H 2 2 soln 
in 250 mL beaker. Neutze to pH 7.5 with ca 0.1 N NaOH, using 
pH meter. Add 10 mL sample from pipet, stir vigorously 1 min, 
and titr. to pH 7.5 with stdzd 0.1/V HCI, using pH meter. 
% NaOH = (mL HCI x normality x 4.0)/(mL sample x sp gr) 

6.116 * Carbon Dioxide— Official Final Action * 

Evolution into std Ba{OH) 2 soln. See 4.15&-4.159, 10th ed. 



6.109 Sulfate Sulfur — Official Final Action 

Slightly acidify soln from 6.106 with HCI (1 +4) and heat to bp. 
Add slowly, with const stirring, slight excess 10% BaCI 2 soln, 
boil 30 min, let stand overnight, and filter. Calc. S from wt 
BaS0 4 , and report as % sulfate S. 

6.110 Total Calcium [27) — Official Final Action 

To 25 mL prepd soln, 6.103(a) or (b), add 10 mL HCI, evap. to 
dryness on steam bath, and H 2 and few mL HCI (1+4), warm 
until all CaCI 2 dissolves, and filter to remove S and any Si0 2 
present. Dil. filtrate to 200-250 mL, heat to bp, add few mL 
NH 4 OH in excess, and then add excess satd (NH 4 ) 2 C 2 A soln. 
Continue boiling until pptd CaC 2 4 assumes well defined gran- 
ular form, let stand 1 hr, filter, and wash few times with hot 
H 2 0. Ignite at 950° in Pt crucible to const wt (CaO) and calc. % 
Ca. CaO x 0.7147 = Ca. 



SODIUM HYPOCHLORITE SOLUTIONS [28) 

Sodium Hypochlorite 

Arsenious Oxide Titration Method— Official Final Action 

6.111 Reagents 

(a) Arsenious oxide std soln. — 0.1/V. Prep, as in 
50.005-50.006. 

(b) Iodine std soln.— Prep, as in 50.018. Stdze against (a). 



6.117 



CALCIUM HYPOCHLORITE AND 
BLEACHING POWDER [28) 

Available Chlorine 

Arsenious Oxide Titration Method 
Official Final Action 



Weigh 5-10 g thoroly mixed sample into porcelain mortar, 
add 30-40 mL H 2 0, and triturate to smooth cream (high-test 
Ca(OCI) 2 will dissolve readily and not form a cream). Add more 
H 2 0, stir well with pestle, and let insol. residue settle few 
moments. Pour mixt. off into 1 L vol. flask, add more H 2 0, and 
thoroly triturate sample and pour off as before. Repeat operation 
until all material is transferred to flask. Rinse mortar and pestle, 
catch wash H 2 in flask, dil. to vol., and mix. Without letting 
material settle, pipet 25-50 mL aliquot into 200 mL erlenmeyer. 
Add excess std As 2 3 soln, 6.111(a), and then decided excess of 
NaHC0 3 . Titr. excess As 2 3 with std I soln, 6.111(b), using starch 
soln, 6.005(f), or I as its own indicator. Subtract vol. I soln, 
corrected to 0.1/V, from vol. As 2 3 soln used, and calc. % 
available CI. 1 mL 0.1/V As 2 3 = 0.003545 g available CI. 



if Surplus method— see inside front cover. 



AOAC Methods (1980) 



Soap 



83 



CHLORAMINE T (28) 

Active Chlorine 

Arsenious Oxide Titration Method — Official Final Action 

6.118 Determination 

Transfer 0.5 g sample to 300-500 mL erlenmeyer, dissolve in 
50 mL H 2 0, and add excess std As 2 3 soln, 6.111(a), and 5 mL 
H 2 S0 4 (1+4). Add decided excess NaHC0 3 and titr. excess As 2 3 
with std I soln, 6.111(b), using starch soln, 6.005(f), or I as its 
own indicator. From this titrn, calc. active CI in sample. 1 mL 
0.1/V As 2 3 = 0.001773 g active CI. (To convert active CI to 
available CI, multiply active CI by 2.) 

6.119 Total Chlorine — Official Final Action 

Dissolve 0.5 g sample in 50 mL H 2 in erlenmeyer and add 
slight excess std As 2 3 soln as calcd from active CI titrn, 6.118. 
Add 5 mL HN0 3 (1+4), neutze with CaCO z , and titr. with std 
AgN0 3 , 50.027-50.029, using K 2 Cr0 4 , 50.028(b), as indicator. Det. 
blank on reagents and correct for any CI found. From corrected 
titrn, calc. % total CI in sample. 1 mL 0.1/V AgN0 3 = 0.003545 
g CI. If total CI exceeds active CI, NaCI is indicated. 

6.120 * Sodium— QfficiaS Final Action * 

From wt sulfated ash. See 6.183, 11th ed. 

MINERAL OILS 

Unsulfonated Residue (30) — Official Final Action 

6.121 Reagent 

(Caution: See 51.030 and 51.031.) 

Fuming 38N sulfuric acid. — In tared g-s bottle (2.5 L acid bottle 
is convenient) mix fuming H 2 S0 4 (free from N oxides) (x) with 
H 2 S0 4 (y) to obtain mixed acid (z), contg slightly >82.38% total 
S0 3 . Depending on strength of fuming acid available, use 
following proportions of 2 acids: 100 partsx (15-20% free S0 3 ) 
to 50 parts/; 100 partsx (20-30% freeS0 3 )to75 parts/; or 100 
partsx (50% free S0 3 ) to 140 parts y. Mix thoroly (considerable 
heat is generated), let cool, and again weigh to det. amt mixed 
acid obtained. Det. exact strength of mixed acid (z) and also of 
reserve supply of acid (y) as follows: 

Pour ca 50 mL into small beaker and fill ca 10 mL weighing 
bulb or pipet by slight suction, wiping off outside of bulb with 
moist, then with dry, cloth. Weigh on analytical balance and let 
acid flow slowly down sides of neck of 1 L vol. flask into ca 200 
mL cold H 2 0. (These sizes of bulb and flask give final soln ca 
0.5/V.) When bulb has drained, wash all traces of acid into flask, 
taking precautions against loss of S0 3 fumes. Dil. to vol. and 
titr. from buret with std alkali, using same indicator as used in 
stdzg. Calc. S0 3 content of both acids, and add calcd amt of 
reserve acid (y) to amt of mixed acid (z) on hand to bring z to 
82.38% total S0 3 (equiv. to 100.92% H 2 S0 4 ). After adding 
required amt of y, again analyze mixed acid to make certain it 
is of proper concn (±0.15% H 2 S0 4 ). Keep acid in small bottles 
or in special dispenser bottle (31) to prevent absorption of H 2 
from air. 

6- 1 22 Determination 

Pipet 5 mL sample into 6" Babcock cream bottle, 16.157(a), 
either 9 g 50% or 18 g 30% type. To reduce viscosity of heavy 
oils, warm pipet after initial drainage by passing it several times 
thru flame; then drain thoroly. If greater accuracy is desired, 
weigh measured sample and calc. exact vol. from wt and sp gr. 

* Surplus method— see inside front cover. 



Slowly add 20 mL 38/V H 2 S0 4 , gently shaking or rotating bottle 
and taking care that temp, does not rise above 60°. Cool in ice- 
H 2 if necessary. When mixt. no longer develops heat on 
shaking, agitate thoroly, place bottle in H 2 bath, and heat 10 
min at 60-65°, keeping contents of bottle thoroly mixed by 
shaking vigorously 20 sec at 2 min intervals. Remove bottle 
from bath and add H 2 S0 4 until oil is in graduated neck. Centrf. 
5 min (or longer if necessary to obtain const vol. of oil) at 
1200-1500 rpm. Read vol. of unsulfonated residue from grad- 
uations on neck of bottle and, to convert to mL, multiply reading 
from 9 g 50% bottle by 0.1 and reading from 18 g 30% bottle 
by 0.2. From result obtained, calc. % by vol. of unsulfonated 
residue. 

SOAP 
Moisture (32) 

6 . 1 23 Toluene Distillation Method — Official Final Action 

Weigh ca 20 g sample into 300-500 mL flask; add 50 mL 
toluene (tech. grade is satisfactory); and, to prevent foaming, 
add ca 10 g lump rosin (do not use powd). Distil into Dean and 
Stark type distg tube receiver and continue distn until no more 
H 2 collects in receiver. Cool contents of tube to room temp., 
read vol. H 2 under toluene in tube, and calc. % H 2 0. 

6.124 * Sodium and Potassium (33) — Official Final Action * 

Removal of metal ions and P, and weighing as chlorides. See 
6.094, 11th ed. 

MINERAL OIL-SOAP EMULSIONS 

Water (34) 

6.125 Toluene Distillation Method — Official Final Action 

Weigh ca 25 g sample and proceed as in 6.123, except use 
less rosin. 

6.126 Total Oil (35)— Official Final Action 

Weigh ca 10 g sample into Babcock cream bottle, 16.157(a). 
Dil. with ca 10 mL hot H 2 and add 5-10 mL H 2 S0 4 (1 + 1). Heat 
in hot H 2 bath ca 5 min to hasten sepn of oil, add enough satd 
NaCI soln to bring oil layer within graduated neck of bottle, 
centrf. 5 min at 1200 rpm, and let cool. Read vol. of oil layer, 
det. density, and from these values calc. wt and %. From this 
% value deduct % fatty acids (and phenols if present), detd sep., 
to obtain % oil. 

6.127 Soap (34)— Official Final Action 

(Error will result if apparent mol. wt of fatty acids varies 
appreciably from that of oleic acid.) 

Weigh 20 g sample into separator, add 60 mL pet ether, and 
ext mixt. once with 20 mL and 4 times with 10 mL 56% alcohol. 
Break emulsion if necessary by letting 1 or 2 mL 20% NaOH 
soln run down wall of separator. Then gently swirl separator 
and let stand few min. Drain ale. layers and wash successively 
thru pet ether contained in 2 other separators. Combine ale. 
exts in beaker and evap. on steam bath to remove alcohol. 
Dissolve residue in ca 100mLH 2 O made alk. with NaOH. Transfer 
to separator, acidify with HCI or H 2 S0 4 , ext 3 times with Et ether, 
and wash ether exts twice with H 2 0. Combine ether exts, evap. 
in weighed beaker on steam bath, and weigh as fatty acids. 
From wt fatty acids, calc. % soap in sample as Na or K oleate. 

6.128 Unsulfonated Residues — Official Final Action 

Using 5 mL of the recovered oil, 6.126, proceed as in 6.122. 



84 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.129 Ash [36)— Official Final Action 

Evap. 10 g sample, or more if necessary, in Pt dish. Ignite, and 
leach charred mass with H 2 0. Ignite residue, add leachings, 
evap. to dryness, ignite, and weigh. From this wt, calc. % ash. 
Test ash for Cu, Ca, CaF 2 , etc. 

ORGANIC MERCURIAL SEED DISINFECTANTS 
Mercury 

6.130 • Volatilization Method [37) * 

Official Final Action 

See 4.150-4.151, 10th ed. {Caution: See 51.041 and 51.065.) 

6.131 * Precipitation Method [37) * 

Official Final Action 

Digestion with H 2 S0 4 and H 2 2 and pptn as HgS. See 6.173, 
11th ed. 

Titrimetric Method [38) — Official Final Action 

6.132 Principle 

Sample is digested under H 2 0-cooled condenser with fuming 
H 2 S0 4 -fuming HN0 3 . Hg is detd by titrn with std SCN soln with 
ferric alum as indicator. Small amts of chloride are oxidized to 
CI and expelled thru condenser. Not applicable in presence of 
large amts of Cl-contg materials. 

6.133 Reagents 

(a) Ferric indicator.— Dissolve 8 g FeNH 4 (S0 4 )2.12H 2 in 80 
mL H 2 0. Add enough HN0 3 to destroy brown Fe color and dil. 
to 100 mL with H 2 0. 

(b) Ferrous sulfate soln. — Dissolve 1 g FeS0 4 .7H 2 in H 2 0, 
add 1 mL H 2 S0 4 , and dil. to 100 mL with H 2 0. Prep, fresh for 
each detn. 

6. 1 34 Preparation of Sample 

(a) Solns. — Mix thoroly and weigh, by difference, amt sample 
(max. 10 g) contg preferably 0.07 g Hg into 500 mLf erlenmeyer. 

(b) Dusts. — Mix thoroly and, using glass weighing dish, weigh 
amt sample as in (a). Transfer thru powder funnel into 500 mL 
f erlenmeyer. 



6.135 



Determination 



(Caution: Conduct detn in well ventilated hood. Method is 

dangerous in presence of material which reacts violently with 

H 2 S0 4 and/or HN0 3 . See 51.019, 51.026, 51.030, 51.031, and 

51.079.) 

Connect straight-tube, H 2 0-cooled condenser to erlenmeyer 
contg sample. Place flask in cold H 2 bath. Carefully add 10 mL 
H 2 S0 4 thru top of condenser and mix by swirling. Add in small 
portions, swirling after each addn, 30-40 mL fuming H^SO* (20% 
free S0 3 ) thru top of condenser, followed by 10 mL red fuming 
HNO z (98% HN0 3 ). Remove from bath and dry outside of flask. 
Heat with small flame to reflux at ca 30 drops/min with red 
fumes persisting in flask and condenser. Heat 30 min; if small 
amt chloride is present, heat 2 hr with occasional addn of fuming 
HN0 3 as required. Cool, and add 100 mL cold H 2 slowly thru 
top of condenser while cooling flask in cold H 2 bath. Add 2 or 
3 glass beads or boiling chips and boil until N oxides have been 
expelled to top of condenser (ca 2 min). Wash condenser with 
50 mL cold H 2 0, disconnect flask, and add satd KMn0 4 soln 

* Surplus method-^see inside front cover. 



until color remains purple. (If large amts insol. material are 
present, filter hot soln thru medium tight asbestos mat in gooch 
before addn of KMn0 4 . Wash flask and filter 5 times with hot 
H 2 0, and then add KMn0 4 .) Cool flask, and destroy KMn0 4 with 
fresh 1% FeS0 4 soln. Add 10 mL ferric indicator and titr. with 
0.1/V NH 4 SCN or KSCN, 50.003-50.004, to appearance of first 
permanent faint orange. 1 mL 0.1/V NH 4 SCN or KSCN - 0.01003 
gHg. 

AOAC-CIPAC Gravimetric Method [39)— -Official Final Action 

(Applicable in presence of large amts Cl-contg materials; not 
applicable to chloro- or nitrophenols nor to materials not de- 
composed by digestion mixt.) 



6.136 



Reagents 



(a) Dilute sulfuric acid.— Add 30 mL H 2 S0 4 to H 2 in 100 mL 
vol. flask, cool, and dil. to vol. with H 2 0. 

(b) Sodium sulfite soln.— 10%. Dissolve 10 g Na 2 S0 3 in H 2 
in 100 mL vol. flask and dil. to vol. with H 2 0. 

(c) Ammonium citrate soln. — pH 7.0. See 2.044(a). 

(d) Precipitating reagent— Add 20 mL 1,2-propanediamine 
(Eastman Kodak Co., P3170) to 100 mL 1/W CuS0 4 soln. Store in 
g-s container. 

(e) Wash soln.— Add 1 g Kl and 2 mL pptg reagent to 1 L H 2 0. 

6.137 Preparation of Sample 

(a) Solns. — Mix thoroly and weigh, by difference, sample 
(max. 5 g) contg 0.02-0.08 g Hg into 125 mL ¥ erlenmeyer. 

(b) Dusts. — Mix thoroly and, using glass weighing dish, weigh 
sample as in (a). Transfer thru powder funnel into 125 mL ¥ 
erlenmeyer. 



6.138 



Determination 



{Caution: Conduct detn in well ventilated hood.) 

Add to sample in following order: 5 mL ethylene glycol, 
swirling to thoroly suspend solids, 4 g Kl, 10 mL dil. H 2 S0 4 , 0.4 
g I, and 2 glass beads. After thoro mixing, connect straight-tube, 
H 2 0-cooled condenser and, with low flame, heat to slight boil 
so that liq. condenses in lower portion of condenser. Swirl 
occasionally, avoiding excessive heat and crystn of large amt I 
in condenser. Reflux 1 hr and, while cooling flask in H 2 bath, 
immediately wash warm condenser with heavy stream of ca 25 
mL H z O. (If dye or I persists in condenser, loosen by reheating 
flask contents, without H 2 in condenser, until liq. refluxes 
slightly beyond adhering material. Wash condenser again with 
ca 25 mL H 2 0, and cool flask.) Disconnect condenser and wash 
connections directly into flask. Add ca 2 mL 10% Na 2 S0 3 
dropwise, with swirling, until I color slightly lightens. (Excess I 
must be present.) Neutze soln with NH 4 OH, using pH test paper, 
until very slightly alk. (pH 7.0-7.3). Cool, and filter with vac. thru 
retentive paper (S&S Blue Ribbon, or equiv.) in buchner into 
400 mL beaker. Wash flask and paper thoroly, keeping total 
filtrate <150 mL. Add 50 mL NH 4 citrate soln, bring mixt. just to 
bp, and stir in 5 mL pptg reagent. Cool and let stand ^2 hr 
(preferably overnight); filterthru medium porosity glass crucible, 
previously dried at 105° and weighed. Transfer ppt with wash 
soln, and wash with same soln several times. Rinse I from ppt 
with ca 25 mL alcohol in 5 mL portions (some samples may 
require up to 50 mL) until filtrate is colorless. (Let alcohol stand 
few min with occasional swirling after each addn before applying 
suction. Ppt should be suspended in liq. each time.) Wash ppt 
with three 5 mL portions CHCI 3 , suspending ppt each time as 
above until dye and pesticides are completely removed. Finally 
wash with 5 mL alcohol, dry 30 min at 105°, cool, and weigh. 

Wt Hg = wt ppt x 0.218. 



AOAC Methods (1980) 



FUMIGANTS 



85 



ANT POISONS AND RODENTBCIDES 

6.139 • Aipha-Naphthylthiourea (40) * 

Official First Action 

{Caution: See 51.039, 51.041, and 51.046.) 
N detn. See 4.132, 10th ed. 

6.140 Thallous Sulfate [41)— Official Final Action 
{Caution: See 51.019, 51.026, 51.031, and 51.041.) 

Weigh sample contg 0.1-0.15 g TI 2 S0 4 (usually 10 g), transfer 
to 800 mL Kjeldahl flask, and add 25 mL H 2 S0 4 followed by 5-10 
ml_ HN0 3 . After first violent reaction ceases, heat until white 
fumes of H 2 S0 4 appear. Add few drops fuming HN0 3 and 
continue heating and adding HN0 3 until org. matter is destroyed, 
as shown by colorless or light yellow solh. Cool, add 10-15 mL 
H 2 0, again cool, and wash contents of flask into 400 mL beaker, 
continuing washing until vol. is 60-70 mL Boil several min to 
remove alt HN0 3 , cool, and filter into 400 mL beaker. Wash with 
hot H 2 until vol. in beaker is 175 mL, neutze with NH 4 OH, and 
then slightly acidify with H 2 S0 4 (1 +4). Add 1 g NaHSO^ to ensure 
reduction of thallic to thallous state. Heat to bp, add 50 mL 70% 
Kl soln, stir,and let stand overnight. Filter thru tight gooch contg 
2 disks S&S 589 white ribbon paper covered by medium pad of 
asbestos. Wash 4 or 5 times with 10 mL portions 1% Kl soln, 
and finally with absolute alcohol. Dry to const wt at 105° (1-1.5 
hr), and weigh as Til. 

% TI 2 S0 4 = (g Til x 0.7619 x 100)/g sample. 



Warfarin (3-(a-Acetonyibenzyl)-4-hydroxycoumarin) (42) 
Official Final Action 

(Applicable to baits contg ca 0.025% and to cones contg ^0.5% 

warfarin. Not applicable to pelleted baits or baits consisting of 

cracked corn treated with ale. warfarin soln and aq. sugar soln, 

and then dried.) 



6.141 



Reagents 



(a) Sodium pyrophosphate soln. — 1%. Dissolve 5 g Na 4 - 
P 2 O 7 .10H 2 O in 500 mL H 2 0. 

(b) Petroleum ether, purified.— Ext 200 mL pet ether with 
three 20 mL portions 1% Na 4 P 2 7 soln. 

(c) Warfarin std soln.— 10 jug/mL Dissolve 100 mg pure 
warfarin (Wisconsin Alumni Research Foundation, P.O. Box 
7365, Madison, Wl 53707) in 100 mL 1% Na 4 P 2 7 soln. Dil. 10 
mL to 100 mL with 1% Na 4 P 2 7 soln, and dil. 10 mL of second 
soln to 100 mL with 1% Na 4 P 2 7 soln. 



6.142 



Determination 



Weigh 10 g sample (0.025%), 0.600 g (0.5%), or equiv. wt of 
higher concn, into 125 mL g-s flask or 100 mL centrf. tube and 
add 50 mL Et ether from pipet. Stopper tightly and shake mech. 
ca 30 min. Transfer 5 or 10 mL to centrf. tube (or centrf. directly), 
stopper, and centrf. 5 min at high speed or until clear. Take 
precautions to avoid evapn of ether. 

Pipet 10 mL 1% Na 4 P 2 7 soln into g-s 16 x 150 mm test tube 
and add 2 mL centrfd ether ext from pipet. Stopper and shake 
vigorously 2 min. Centrf. at high speed until aq. layer is clear. 
Draw off ether layer, including any emulsion that remains, using 
fine-tip glass tube attached to aspirator. Add ca 2 mL Et ether, 
shake vigorously, centrf., and completely draw off ether layer. 
Repeat ether extn, and then ext twice with purified pet ether in 
same manner. 

Prep, blank soln similarly, using 2 mL ether instead of 2 mL 
ether ext. 



Del A of aq. soln in 1 cm silica cell at 308 nm against blank 
soln in Beckman spectrophtr, model DU (replaced by models 
24/25), or equiv. Uet.A' (ca 0.46) of the std warfarin soln against 
1% Na 4 P 2 7 soln. 
% Warfarin - (A/A') x (10" 5 g std/mL) 

x [100/(g sample x (2/50)0/10))] 
= (A/ A') x (0.250/g sample). 

FUMIGANTS 

Fumigant Mixtures (43) — Official First Action 

(Applicable to org. components of CS 2 , CCI 4 , (CH 2 ) 2 CI 2 , and 
(CH 2 ) 2 Br 2 mixts. Precautions: Handle with care in hood or well- 
ventilated area. Mixts are volatile, poisonous, and sometimes 
flammable and may be fatal if inhaled or swallowed. They cause 
skin and eye irritation. In case of contact, immediately remove 
contaminated clothing and flush affected area with copious 
amts of H 2 0. Do not reuse clothing until free of contamination. 
Do not use containers or equipment of At, Mg, or their alloys.) 

6.143 Principle 

Components are detd by GLC. Peak area of each component 
is measured and compared to stds of same fumigant mixt. 
Precision of method is ±0.6% for each component. 

6.144 Sampling 

Obtain representative 1 L sample from container. Sample bulk 
containers by means of weighted bottle, lowered toward bottom 
and raised at such rate that it is % full when withdrawn. Sample 
drums or small containers with thief or thru tap or valve located 
so that sample comes from well below surface. Prevent contam- 
ination of product or sample. 

Place sample in clean, dry, and solv. vapor-tight glass bottle 
of such size that it is nearly filled {not above shoulder) by 
sample. Vapor-tight g-s bottles or screw-cap bottles with Sn foil 
lined caps are satisfactory. Store samples at low temp.; cool to 
<18° before opening for analysis. 

6.145 Apparatus 

(a) Gas chromatograph. — With flame ionization or thermal 
conductivity detector. Typical operating conditions: Column 
temp., 110°; injection port temp., 200°; flow rate, 80 mL He/ min. 

(b) Recorder. — 0.05-1.05 mv, full scale response. Integrator 
may be used. 

(c) Syringe. — Hamilton Co. 10 /jlL No. 701 N, or equiv. 

(d) Column.— -1.2 m (4') stainless steel, V od, 0.194" id, 
packed with reagent 6.146(a). Max. temp, is 160°. Other columns 
can be used but chromatgc conditions and sample size must be 
adjusted in accordance with column requirements. One such 
column is: 3 m (10') stainless, 3 /ie"od, 0.12" id, packed with 20% 
by wt /V,/V-bis-(2-cyanoethyl) formamide on 80-100 mesh Chro- 
mosorb W, acid-washed. Columns are available from com. 
suppliers. Criterion for use is emergence of each component of 
mixt. of CS 2 , CCI 4 , {CH 2 ) 2 CI 2 , and (CH 2 ) 2 Br 2 as sep. peak. 



6.146 



Reagents 

-30% by wt tricresyl phosphate on Chro- 



(a) Column packing.- 
mosorb P, 30-60 mesh. 

(b) Carbon disulfide std.— ACS. 

(c) Carbon tetrachloride std. — ACS. 

(d) Ethylene d /'chloride std. — Purified 1,2-dichloroethane, 
available from laboratory supply houses, or use center cut of 
fractionation of com. product. 

(e) Ethylene dibromide {1,2-dibromoethane) std. — Purified or 
distd as in (d). 



86 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.147 Preparation of Standards 

Prep, fresh stds just before analysis which approximate ex- 
pected composition, by wt, of fumigant mixt. Place proper wt 
of each component in 25 mL g-s vol. flask and mix well. Do not 
prep, by vol. Cool CS 2 to prevent loss. Adjust wt stds to detector 
response. 

Carefully fill weighed 10 mL vol. flask to mark with prepd std 
and weigh. Use this wt to det. g/5 /xL values for each component 
of std. 



6.148 



Determination 



Purge column thoroly at 1 10° before use. Establish baseline 
at full sensitivity. Inject 5 /xL std fumigant mixt. into chromato- 
graph. Attenuate successively so that each peak is at max. % of 
chart scale, adjusting sample size and attenuation, if necessary. 
Repeat injection, Detd area for each component, corrected for 
any baseline drift, should differ by ^1%. Order of elution from 
column is: CS 2 , CCI 4 , (CH 2 ) 2 CI 2 , and (CH 2 ) 2 Br 2 . Total analysis time 
is ca 21 min. 

Inject 5 ^1 sample into chromatograph. Det. corrected area of 
each component from chromatogram, or note integrator read- 
ing. 

g Component = Sx C/B, where S - wt component in std, 
B = area for component in std, and C = area for component in 
sample. Perform calcn for each component in sample. 

% Component = g component in sample x 100/ sum of g 
components in sample. 

Last equation is not applicable in presence of unmeasured 
contaminants. 



PESTICIDES RELATED TO NATURAL 
PRODUCTS AND THESR SYNERGISTS 

Technical AlBethrin [44) — Official First Action 

{Caution: See 51.041.) 
6.149 Principle 

Allethrin reacts quant, with ethylenediamine to form chrysan- 
themum monocarboxylic acid which is detd by titrn with std 
NaOMe in pyridine. Chrysanthemum monocarboxylic acid, an- 
hydride, and acid chloride interfere quant, and are detd inde- 
pendently. 



6.150 



Reagents 



(a) Absolute alcohol. — SDF No. 2-B is satisfactory. 

(b) Methanol ic hydrochloric acid std soln. — 0.1 N. Dil. 17 mL 
HCI (1 + 1) to 1 L with anhyd. MeOH. Stdze against std 0.1/V 
NaOH, using phthln. If used at temp., T, different from that at 
which stdzd, T , calc. corrected normality = /V[1-O.001{r - T )]. 

(c) Sodium methy late std soln. — 0.1/V in pyridine. Transfer 50 
mL 2/V NaOMe {Caution: See 51.038) to 1 L bottle contg 75 mL 
anhyd. MeOH and dil. to 1 L with redistd pyridine. Stdze against 
NBS benzoic acid, using pyridine as solv. and thymolphthalein, 
(i), as indicator. Dispense from 50 mL automatic buret with vents 
connected to Ascarite tubes. Stdze daily against std methanolic 
HCI, (b). 

(d) Methanolic potassium hydroxide std soln. — 0.02/V. Dis- 
solve 1.12 g KOH in 1 L MeOH. Stdze as in 50.035. 

(e) Morphoiine soln.— Transfer 8.7 mL redistd morpholine to 
1 L bottle and dil. to 1 L with anhyd. MeOH. Fit bottle with 2- 
hole rubber stopper; thru 1 hole insert 20 mL pipet so that tip 
extends below surface of liq., and thru other hole insert short 
piece of glass tubing to which is attached aspirator bulb. 

(f) Ethylenediamine.— Redistd com. grade contg <3% H 2 0. 



Dispense from automatic buret with vents connected to Ascarite 
tubes. 

(g) Dimethyl yellow-methylene blue mixed indicator. — Dis- 
solve 1 g dimethyl yellow (p-dimethylaminoazobenzene; Cau- 
tion: See 51.085) and 0.1 g methylene blue in 125 mL anhyd. 
MeOH. 

(h) a-Naphtholbenzein indicator. — 1% ale. soln. 

{ i) Thymolphthalein in dicator. — 1% pyridinesoln. 

6.151 Determination of Chrysanthemum 

Monocarboxylic Acid Chloride 

Add 8-10 drops mixed indicator, (g), to ca 150 mL anhyd. 
MeOH and add 0.1/V HCI, (b), dropwise until soln appears reddish 
brown by transmitted light. Add 0.02/V KOH, (d), dropwise until 
appearance of first green. Transfer 25 mL to each of three 125 
mL g-s erlenmeyers, reserving 1 flask as ref. color for end point. 
Into each of other flasks add 1.5-2.5 g sample from weighing 
pipet, swirling flask while adding sample. Within 5 min, titr. with 
0.02/V KOH, (d), to first green end point, using blank as ref. 
color. Calc. milliequiv. chrysanthemum monocarboxylic acid 
chloride/g sample, C = V x /V/g sample, where V= mL N 
normal KOH required; 
% Chrysanthemum monocarboxylic acid chloride = Cx 18.67. 

6.152 Determination of Chrysanthemum 

Monocarboxylic Acid 

Transfer 25 mL anhyd. alcohol to each of two 125 mL g-s 
erlenmeyers, add 8-9 drops a-naphthoibenzein indicator, and 
cool to 0° in ice bath. Neutze by adding 0.02/V NaOH dropwise 
to bright green end point. To each flask add 1.5-2.5 g sample 
from weighing pipet. Immediately titr. with 0.02/V NaOH, 50.034, 
to first bright green end point. Calc. milliequiv. chrysanthemum 
monocarboxylic acid and acid chloride/g sample: D = Xx A//g 
sample, where X = mL N normal NaOH required; {D ~ C) x 
16.82 = % chrysanthemum monocarboxylic acid. 

6.153 Determination of Chrysanthemum 

Monocarboxylic Anhydride 

Pipet 20 mL morpholine soln, (e), into each of four 250 mL 
erlenmeyers, using same pipet. Fill pipet by exerting pressure 
in bottle with aspirator bulb. Reserve 2 flasks for blanks; into 
each of other flasks add 1.5-2.5 g sample from weighing pipet. 
Swirl flasks and let samples and blanks stand 5 min at room 
temp. Add 4-5 drops mixed indicator, (g), to each flask and titr. 
with 0.1/V HCI, (b), until color changes from green to faint red 
when viewed by transmitted light. Calc. milliequiv. chrysanthe- 
mum monocarboxylic anhydride/g sample: E = (B - Y) x /V/g 
sample, where Y = mL N normal HCI required for sample, and 
B = mL/V normal HCI required for blank; [E - 2C) x 31.84 = 
% chrysanthemum monocarboxylic anhydride. 



6.154 



Determination of Allethrin 



Add sample contg 0.8-1.1 g allethrin to each of two 250 mL 
erlenmeyers from weighing pipet. To each of 2 flasks as blanks 
and to samples add 25 mL ethylenediamine, (f), with swirling. 
Let samples and blanks stand 2 hr at 25±2°. Wash down sides 
of flasks with 50 mL redistd pyridine. To each flask add 6-10 
drops thymolphthalein indicator, (i), and titr. with 0.1/V NaOMe, 
(c), to first permanent blue-green end point. (With colorless 
samples, first blue end point may be used.) Calc. milliequiv. 
allethrin/g sample: F ~ (Z - B) x/V/g sample, where Z = mL 
N normal NaOMe required for sample, and B — av. mL N nor- 
mal NaOMe required for blank; (F + C - D - E) x 30.24 = % 
allethrin. 



AOAC Methods (1980) 



Natural Products 



87 



d-trans- AWethr'm (rf/-2- Ally 1-4- hydroxy-3-methy 1-2- 

cyclopentene-1 Ester of o*-fra/?s-2,2-Dimethyl-3- 

(2-methylpropenyl)cyclopropanecarboxylic Acid) (45) 

Official Final Action 

Gas Chromatographic Method 

{Caution: See 51.041.) 
6.155 Principle 

</~?ra/7s-Allethrin is dild in acetone contg dibutyl phthalate as 
internal std. Ratios of GLC peak hts of tf-fra/?s-allethrin and 
dibutyl phthalate in sample and std are compared for quant, 
detn. Method is applicable to both tech. tf-frans-allethrin and 
various formulations of it. Not applicable to formulations contg 
large amt MGK Repellent 874 (2-hydroxyethyl-A7-octyl sulfide). 



6.156 



Apparatus and Reagents 



(a) Gas chromatograph. — Equipped with flame ionization de- 
tector and 1.2 m (4') x 4 mm id glass column packed with 5% 
OV-1 (Analabs, Inc.) on 80-100 mesh Chromosorb W (HP). 
Operating conditions: temps (°) — column 165, injection port 230, 
detector 230; gas flows (mL/min) — N carrier gas 125, air 350—400, 
H 40-50; sensitivity — 10~ 9 amp full scale, attenuation 4x for 
tech. material, 10~ 9 amp full scale, attenuation 1 for formulations. 
Before use, condition column 2-3 hr at 275° with N flow 50 
mL/min. If necessary, vary column temp, or gas flow to attain 
retention times of ca 4 and 7 mi n for internal std and d-trans- 
allethrin, resp. Also vary detector sensitivity or injection vol. to 
attain =*100 mm peak ht for each compd {ca 16 yag d-trans- 
allethrin). Theoretical plates/ft must be >200. 

Calc. theoretical plates/ft (/V) as follows: N = 16L 2 /(M Z x F), 
where L = retention of GLC peak in mm; M - peak baseline 
produced by drawing tangents to points of inflection of peak; 
and F - length of column (ft). 

(b) Internal std soln. — 4.0 mg dibutyl phthalate/mL acetone. 

(c) d-trans-Allethrin std solns. — ( 1 ) Soln 1. — Approx. 4 
mg/mL. Accurately weigh ca 1.0 g d-trans- aWethnn (available 
from McLaughlin Gormley King Co., 8810 Tenth Ave N, Min- 
neapolis, MN 55427) into 50 mL vol. flask and dil. to vol. with 
acetone. Pipet 20 mL this soin into 100 mL vol. flask, add 50 mL 
internal std soln by pipet, and dil. to vol. with acetone. Use this 
soln for detn of tech. material. (2) Soln 2. — Approx. 1 mg/mL. 
Pipet 25 mL Soln 1 into 100 mL vol. flask and dil. to vol. with 
acetone. Use this soln for detn of d-trans-a\\e\hnx\ in formula- 
tions. 

6.157 Preparation of Sample 

(a) Technical material. — Accurately weigh sample contg ca 
1.0 g cf-frans-allethrin into 50 mL vol. flask and dil. to vol. with 
acetone. Pipet 20 mL aliquot into 100 mL vol. flask, add 50 mL 
internal std soln by pipet, and dil. to vol. with acetone. 

(b) Formulations. — Accurately weigh sample contg ca 200 mg 
cZ-frans-allethrin into 50 mL vol. flask, add 25 mL internal std 
soln by pipet, and dil. to vol. with acetone. Pipet 25 mL aliquot 
into 100 mL vol. flask and dil. to vol. with acetone. 

6.158 Gas Chromatography 

(a) Technical material. — Inject altquots (ca 3 fxl) std Soln 1 
until ratio of c/- fra/?s-a I let hrin: dibutyl phthalate peak hts varies 
<1% for successive injections. Repeat with sample soln, fol- 
lowed by duplicate injections of std soln. If peak ht ratios differ 
>±1% from previous std injections, repeat series of injections. 

(b) Formulations. — Proceed as in (a), using std Soln 2. Repeat 
std injections after each series of 3 sample injections. If peak ht 
ratios differ >±1.5% from previous std injections, repeat injec- 
tions. 



6.159 Calculations 

(a) Technical material. — Calc. peak ht ratios for duplicate std 
injections before and after sample injections and average the 4 
values. Calc. and average peak ht ratios for sample injections. 

% c/-fr3A?s-Allethrin = (^'xPx R)/{W x /?'), 
where W and IV = g std and sample, resp.; P ~ % purity of std; 
and R' and R = peak ht ratios of std and sample, resp. 

(b) Formulations. — Calc. av. for all std peak ht ratios and for 
sample peak ht ratios. 

% cT-f/ans-Allethrin = (W x P x R x 2)/{W x /?'), 
where W - g std in final diln. 



DERRIS AND CUBE POWDER 

Rotenone 

Crystallization Method [46)— Official Pinal Action 

(Caution: See 51.049 and 51.084.) 

6.160 Reagents 

(a) Purified rotenone. — Dissolve rotenone in boiling CCI 4 ; cool 
in refrigerator or ice bath at 0-10° until pptn of rotenone-CCI 4 
solvate stops. Filter thru buchner and wash once or twice with 
ice-cold CCI 4 . Cone, filtrate, crystallize, and filter as before. 
Transfer cryst. residue to beaker, add ca twice their vol. alcohol, 
and heat nearly to boiling. (Crystals need not dissolve com- 
pletely.) Cool to room temp., filter thru buchner, and draw air 
thru cryst. residue until most alcohol is removed. Remove 
rotenone from funnel, dry in air, and finally heat 1 hr at 105°. 
Mp, detd in Pyrex, of purified material should be 163-164°. 
(Mother liquors may be coned and rotenone-CCI 4 solvate allowed 
to crystallize. Cryst. material may be used for further purification, 
or kept for prepn of wash solns or for seeding to induce crystn 
in detn.) 

(b) Rotenone-CCI 4 solvate.— Ppt rotenone from CCI 4 soln, 
filter by suction, and dry in air. 

(c) Rotenone-CCi 4 wash soln.— Sat. CCI 4 at 0°, and keep at 0° 
during use. 

(d) Alcohol saturated with rotenone at room temp. 

(e) Charcoal, activated. — Norit-A neutral, or equiv. 

6.161 Preparation of Solution 

(a) Weigh 30 g (if sample contains >7% rotenone, use amt 
to give 1.0-1.5 g rotenone in 200 mL aliquot) finely powd root 
and 10 g of the C, (e), into 500 mL g-s erlenmeyer. Add 300 mL 
CHCI3, measured at known room temp.; fasten stopper securely 
and place flask on shaking machine. Agitate vigorously 5*4 hr, 
preferably interrupting shaking with overnight rest (or flask may 
be shaken continuously overnight). Rapidly filter mixt. into 
suitable flask, using fluted paper without suction and keeping 
funnel covered with watch glass to avoid evapn loss. Stopper 
flask and adjust temp, of filtrate to that of original CHCI 3 . 

(b) Alternative extraction method. — If sample has ratio of 
rotenone to total ext of >0.4, use amt contg 1.0-1.5 g rotenone 
and successively ext 4 times with CHCI 3 , using 300 mL CHCI 3 
and 4 hr agitation for first extn as in (a) and 200 mL and 2 hr 
each for other extns. Filter after each extn and return marc to 
flask for extn with fresh solv. Finally combine exts, evap. almost 
to dryness, and use entire ext to det. rotenone. 

(c) Extraction method for formulations containing 0.75-1.0% 
rotenone with or without sulfur and /or pyrethrins. — Weigh two 
50 g portions sample into sep. 500 mL g-s erlenmeyers. Add 5 
g of the C and 300 mL CHCI 3 , measured at known room temp., 
to each. Stopper and continue as in (a). 



88 



6. Pesticide Formulations 



AOAC Methods (1980) 



6. 1 62 Determination 

{Caution: See 51.011, 51.018, 51.046, 51.049, and 51.056.) 

Pipet 200 mL soln, 6.161 (or entire soln if alternative extn, (b), 
is used), into 500 mL Pyrex erlenmeyer and distil until ca 25 mL 
remains. (For formulations, 6.161(c): In absence of S, combine 
the 2 exts in one of the erlenmeyers. In presence of S, remove 
all CHCI 3 on steam bath in air current, avoiding prolonged 
heating. Add 35 mL acetone to each residue and boil gently on 
steam bath to dissolve all resins. Remove from steam bath, 
stopper tightly, and hold 2 hr at 0-5°. Filter both acetone solns 
thru same 15 mL, medium porosity, fritted glass buchner into 
single 500 mL erlenmeyer. Rinse and wash with acetone at 5°. 
Remove acetone as CHCI 3 was removed above.) 

Evap. almost to dryness on steam bath in current of air. 
Remove remainder of solv. under reduced pressure, heating 
cautiously on steam bath when necessary to hasten evapn. 
(Suction may be applied directly to flask if stopper with vent is 
used to release pressure, so that excessive vac. may be avoided. 
Use flasks with slightly convex bottoms; do not use flasks below 
av. wt.) Dissolve ext in 15 mL hot CCI 4 and again, in similar 
manner, remove all solv. Repeat with another 10-15 mL portion 
hot CCI4. (This treatment removes all CHCI 3 from resins. CHCI 3 
ext is usually completely sol. in CCI 4 ; if small amts of insol. 
material are present, purification described later will eliminate 
them.) 

Dissolve residue in ca 10 mL CCI 4 and transfer quant, with hot 
CCI4 to 50 mL erlenmeyer marked at 25 mL. Adjust vol. to 25 mL 
by evapg on steam bath or by adding CCI 4 . Cool flask in ice bath 
several min, stopper flask, and swirl until crystn is apparent. 
Seed with few crystals of rotenone-CCI 4 solvate if necessary to 
induce crystn. If at this stage only small amt of cryst. material 
seps, add accurately weighed amt of purified rotenone, 6.160(a), 
estd to be enough to assure that final result, expressed as pure 
rotenone, is sM g. Then warm to dissolve completely, and again 
induce crystn. At same time prep, satd soln of rotenone in CCI 4 , 
6.160(c), for washing. Place flasks contg ext and washing soln 
in ice bath capable of holding temp, at 0°, and let stand overnight. 
(Store ice bath in refrigerator to keep ice from melting too 
rapidly.) 

After 17-18 hr in ice bath, rapidly filter ext thru weighed gooch 
fitted with filter paper disk, removing flask from ice bath only 
long enough to pour each fraction of ext into crucible. Rinse 
cryst. residue from flask and wash under suction once with the 
ice-cold satd rotenone-CCI 4 wash soln. (=^12-15 mL soln should 
be used for rinsing and washing.) Continue suction ca 5 min; 
then dry to const wt at 40° (ca 1 hr). Wt obtained is crude 
rotenone-CCI 4 solvate. 

Break up contents of crucible with spatula, mix thoroly, and 
weigh 1.000 g into 50 mL erlenmeyer. Add 10 mL alcohol 
previously satd with rotenone at room temp., swirl flask few 
min, stopper tightly, and set aside ^4 hr, preferably overnight, 
at same temp. Filter on weighed gooch fitted with filter paper 
disk. Rinse crystals from flask and wash under suction with 
alcohol satd with rotenone at temp, of recrystn (ca 10 mL usually 
required). Continue suction 3-5 min and then dry crucible at 
105° to const wt (ca 1 hr). 

Multiply g residue by g total crude rotenone-CCt 4 solvate, and 
add 0.07 g to product as correction for rotenone held in soln in 
the 25 mL CCI 4 used in crystn. If any pure rotenone was added, 
subtract its wt from value obtained. This gives wt pure rotenone 
contained in aliquot of ext. 

Note: Most important precaution in using this method is to 
keep temp, of CCl 4 -rotenone wash soln and crucibles as near 0° 
as possible. Keep wash soln surrounded by crushed ice except 
when actually being used. In warm weather keep crucibles in 
refrigerator until ready to use. 



Infrared Method {47) — Official First Action 

(Not applicable to derris products) 



6.163 



Standardization 



Prep, std solns of purified rotenone, 6.160(a), in CHCI 3 at 
concns of 5, 10, 15, and 20 mg/mL. Scan each std soln from 7.0 
to 8.0 pirn at speed of 6 min/jLim and scale of 10 cm/jum, using 
0.1 mm cell and accurately matching cell filled with CHCI 3 as ref. 
Scan each in duplicate. Obtain av. A of each concn, using 7.57 
)Ltm as base point and 7.65 /im as peak. Plot A against concn. 



6.164 



Determination 



Weigh sample contg 250-300 mg rotenone into 25 x 200 mm 
culture tube. Add 1-2 g anhyd. Na 2 S0 4 , 2 g activated charcoal, 
and 50 mL CHCI 3 by pipet. Close securely with Teflon-lined 
screw cap and tumble end over end 1 hr at ca 35 rpm. Filter thru 
medium paper, avoiding evapn losses. Transfer 20 mL aliquot 
to 50 mL erlenmeyer and evap. on steam bath with current of 
air. Transfer residue to 10 mL g-s vol. flask and dil. to vol. with 
CHCI 3 . Stopper, and mix thoroly. 

Scan from 7.0 to 8.0 /im, using 0.1 mm cell and matched cell 
filled with CHCI 3 as ref. Det.4 by baseline method from 7.57 to 
7.75 fxm and peak at 7.65 ptm, using same scanning speed and 
scale expansion as in stdzn. 

Calc. % rotenone from std curve and wt sample in final diln. 

6.165 Total Ether Extract— Official Final Action 

{Caution: See 51.009, 51.039, 51.054, and 51.070(b).) 

Ext 5 g finely powd root with ether 48 hr in Soxhlet or other 
efficient extn app. Cone, ext and filter off any insol. material 
present. Receive filtrate in tared beaker, evap. ether on steam 
bath, and dry in oven at 105° to const wt. 

Piperonyl Butoxide [48)— Official Final Action 

6.166 Apparatus and Reagents 

(a) Photoelectric colorimeter. — Equipped with narrow band- 
pass interference type filter with central wavelength 630 nm. 
(Filter is available from: Baird-Atomic Inc.; Bausch and Lomb 
Optical Co., 10 Champeney Terrace, Rochester, NY 14602; and 
Photovolt Corp., 1115 Broadway, New York, NY 10010.) Spec- 
trophtr set at wavelength in range 625-635 nm may also be 
used. 

(b) Purified tannic acid. — Purify as follows: To 20 g tannic 
acid (USP reagent grade) add 100 mL EtOAc (99%) and stir 
mech. ca 1 hr. Filter by suction thru fritted glass funnel, and 
wash residue with three 5 mL portions EtOAc. To combined 
filtrate and washings add 2 g finely powd Darco G-60 (or equiv. 
decolorizing C), and stir mech. ca 0.5 hr. Filter by gravity thru 
double thickness Whatman No. 1 , or equiv., paper into graduated 
dropping funnel. Wash residue several times with EtOAc until 
vol. filtrate and washings is ca 125 mL. Place dropping funnel 
over 1 L, 3-neck, r-b flask, equipped with mech. stirrer, and with 
vigorous agitation in flask, add filtrate dropwise to 5 times its 
vol. of toluene. Purified tannic acid is pptd immediately. 

Filter by suction thru fritted glass funnel, and wash product 
thoroly with toluene, stirring solids with toluene to assure 
complete removal of EtOAc. Continue suction until practically 
alt toluene is removed. Dry purified tannic acid in vac. oven at 
ca 40°, and place in tightly stoppered bottle. 

(c) Tannic acid reagent. — Completely dissolve exactly 0.025 
g purified tannic acid in 20 mL HOAc by shaking at room temp. 
Add 80 mL H 3 P0 4 and mix thoroly. Prep, fresh daily. Store tightly 
stoppered, as it is hygroscopic. 



AOAC Methods (1980) 



Natural Products 



89 



(d) Purified piperonyl butoxide. — Purify by low pressure frac- 
tional distn of tech. product. (Caution: See 51.015.) Also available 
from Fairfield American Corp., 3932 Salt Rd, Medina, NY 14103. 

(e) Piperonyl butoxide std sofn.— SO fjiQ/QA mL Weigh exactly 
1.000 g purified piperonyl butoxide into 100 mL vol. flask. 
(Hypodermic syringe and needle are convenient for adding 
compd to flask.) Dil. to vol. with deodorized kerosene and mix 
well. Pipet 10 mL of this soln into 200 mL vol. flask. Dil. to vol. 
with deodorized kerosene and mix well. This soln is stable for 
several months. If std is to be used with sample contg pyrethrum, 
add enough pyrethrum ext to std before initial diln to give ratio 
piperonyl butoxide to pyrethrins similar to sample. 

6.167 Preparation of Sample 

Accurately weigh sample contg 0.5-1.5 g piperonyl butoxide 
into tared 100 mL vol. flask, dil. to vol. with deodorized kerosene, 
and mix well. Pipet 10 mL into 200 mL vol. flask, dil. to vol. with 
deodorized kerosene, and mix well. 



6.168 



Determination 



Pipet 0.1 mL (from 1 mL pipet graduated in 0.1 mL) sample 
soln into 18 x 150 mm test tube. Add exactly 5 mL tannic acid 
reagent and shake vigorously 1 min. Treat std and blank, 
consisting of 0.1 mL deodorized kerosene, simultaneously in 
same manner. 

Place test tubes in test-tube basket and place in vigorously 
boiling H 2 bath 5 min. Remove basket and let tubes cool to 
room temp. Transfer solns to colorimeter tubes and read, against 
H 2 0, using 625-635 nm filter or setting. (After cooling to room 
temp, there is no appreciable change in A for several hr.) 

Subtract A Q of deodorized kerosene from readings of both 
sample, A, and std, A\ 

mg Piperonyl butoxide = A x 0.05/>4\ 



PYRETHRINS 
Mercury Reduction Method (49) — Official Final Action 

(Caution: See 51.039, 51.054, 51.070, and 51.073.) 

6.169 Reagents 

(a) Deniges reagent — Mix 5 g yellow HgO with 40 mL H 2 0, 
and, while stirring, slowly add 20 mL H 2 S0 4 ; then add addnl 40 
mL H 2 and stir until all dissolves. Test for absence of mercurous 
Hg by adding few drops of (b) to 10 mL and titrg with (c) as in 
6.171, par. 2, beginning "Add 50 mL previously prepd and 
cooled dil. HCI . . ." 

(b) Iodine monochloride soln. — Dissolve 10 g K! and 6.44 g 
KI0 3 in 75 mL H 2 in g-s bottle; add 75 mL HCI and 5 mL CHCI 3 , 
and adjust to faint I color (in CHCI 3 ) by adding dil. Kl or KI0 3 
soln. If much I is liberated, use stronger soln of KI0 3 than 0.01/W 
at first, making final adjustment with 0.01M soln. Keep in dark 
and readjust when necessary. Do not store in refrigerator. 

(c) Potassium iodate std soln. — 0.01M Dissolve 2.14 g pure 
KI0 3 , previously dried at 105°, in H 2 and dil. to 1 L 1 mL = 0.0057 
g pyrethrin I and needs no further stdzn. 

(d) Alcoholic sodium hydroxide soln. — (1) 1.0N. — Dissolve 40 
g NaOH in alcohol and dil. to 1 L with alcohol. (2) 0.5N—OW. 
1.0A/ with alcohol (1 + 1). 

(e) Petroleum ether. — Aromatic-free, bp range 30-60°. 

(f) Ethyl ether. — Peroxide-free, reagent grade. 

6.170 Preparation of Sample 

(a) Pyrethrum powder. — Ext sample contg 40-150 mg total 
pyrethrins in Soxhlet or other efficient extn app. 7 hr with pet 



ether. After extn is complete, evap. pet ether to ca 40 mL, 
stopper flask, and place in refrigerator at 0±0.5° overnight. Filter 
cold ext thru cotton plug satd with cold pet ether, in stem of 
funnel, collecting filtrate in 250 mL erlenmeyer. Wash with three 
15 mL portions cold pet ether. Evap. filtrate and washings on 
H 2 bath, using air current, until <1 mL solv. remains. 

Add 15-20 mL 0.5/V ale. NaOH to evapd ext, connect to reflux 
condenser, and boil gently 1-1.5 hr. Transfer to 600 mL beaker 
and add enough H 2 to bring vol. to 200 mL. Add few glass 
beads, or preferably use boiling tube, and boil down to 150 mL. 
Transfer to 250 mL vol. flask and add 1 g Fiiter-Cel and 10 mL 
10% BaCI 2 soln. Do not shake before dilg to vol. Dil. to vol., mix 
thoroly, filter off 200 mL, neutze with H 2 S0 4 (1 +4), using 1 drop 
phthln, and add 1 mL excess. (If necessary to hold soln overnight 
at this point, leave in alk. condition.) 

(b) Pyrethrum extracts in mineral oil. — Weigh or measure 
sample contg 40-150 mg total pyrethrins, add 50 mL pet ether 
and 1 g Filter-Cel, and place in refrigerator at 0±0.5° overnight. 
Filter thru gooch into 300 mL erlenmeyer and wash with three 
15 mL portions cold pet ether. Evap. filtrate and washings on 
H 2 bath, using air current, until <1 mL solv. remains. 

Add 20 mL 1/V ale. NaOH, or more if necessary, to ext, connect 
to reflux condenser, and boil gently 1-1 .5 hr. Transfer to 600 mL 
beaker and add enough H 2 to make aq. layer 200 mL. If >20 
mL ale. NaOH soln was used, add enough H 2 so that all alcohol 
is removed when vol. is reduced to 150 mL. Add few glass 
beads, or preferably use boiling tube, and boil aq. layer down 
to 150 mL. Transfer to 500 mL separator and drain aq. layer into 
250 mL vol. flask. Wash oil layer once with H 2 and add wash 
H 2 to aq. portion. If slight emulsion still persists after draining 
aq. layer and washings, add 2-3 mL 10% BaCI 2 soln, but do not 
shake vigorously after adding BaCl 2 because reversed emulsion 
difficult to sep. may form. To aq. soln in 250 mL flask add 1 g 
Filter-Cel and ^10 mL of the BaCI 2 soln. Swirl gently and let 
stand 30 min. Dil. to vol., mix thoroly, and filter off 200 mL. Test 
filtrate with BaCI 2 soln to see if enough has been added to obtain 
clear soln. Neutze with H 2 S0 4 (1+4), using 1 drop phthln, and 
add 1 mL excess. (If necessary to hold soln overnight at this 
point, leave in alk. condition.) 

6.171 Determination of Pyrethrin I 

Filter acid soln from 6.170(a) or (b) thru 7 cm paper, coated 
lightly with suspension of Filter-Cel in H 2 0, on buchner, and 
wash with three 15 mL portions H 2 0. Transfer to 500 mL g-s 
separator and ext with two 50 mL portions pet ether. Shake 
each ext s*1 min, releasing pressure if necessary by inverting 
separator and carefully venting thru stopcock. Let layers sep. 
^5 min or until aq. layer is clear before draining and re-extn. 
Reserve aq. layer for pyrethrin II detn. Do not combine pet ether 
exts but wash each in sequence with same three 10 mL portions 
H 2 0, and filter pet ether exts thru small cotton plug into clean 
250 mL separator. Wash separators and cotton in sequence with 
5 mL pet ether. Ext combined pet ether solns with 5 mL 0.1/V 
NaOH, shaking vigorously 5=1 min. Let layers sep. ^5 min before 
draining aq. layer into 100 mL beaker. Wash pet ether with addnl 
5 mL portion 0.1/V NaOH and with 5 mL H 2 0, adding washings 
to beaker. Add 10 mL Deniges reagent and let stand in complete 
darkness 1 hr at 25±2°. 

Add 20 mL alcohol and ppt HgCI with 3 mL satd NaCI soln. 
Warm to ca 60° and let stand several min until ppt coagulates 
and settles. Filter thru small paper, transferring all ppt to paper, 
and wash with sMO mL hot alcohol. Wash with 2 or more 10 mL 
portions hot CHCI 3 and place paper and contents in 250 mL 
g-s erlenmeyer. Add 50 mL previously prepd and cooled dil. HCI 
(3+2). Add 5 mL CHCI 3 or CCI 4 and 1 mL freshly adjusted ICI 



90 



6. Pesticide Formulations 



AOAC Methods (1980) 



soln, and titr. with 0.1/W KI0 3 soln, shaking vigorously ^30 sec 
after each addn, until no I color remains in CHCI 3 or CCI 4 layer. 
Take as end point when red color disappears from solv. layer 
and does not return within 1-3 min. From mL std Kt0 3 soln used 
in titrn and blank on Deniges reagent, calc. % pyrethrin I. 

(Reactions: 

2Hg 2 CI 2 + 4ICI = 4HgCI 2 + 2I 2 
2I 2 + KI0 3 + 6HCI = KCI + 5ICI + 3H 2 
Addn of ICI does not change vol. relationship between mer- 
curous Hg and KI0 3 soln, and aids in detg end point in titrn of 
small amts of Hg.) 

Note: Chrysanthemum monocarboxylic acid reacts with De- 
niges reagent to form series of colors beginning with phthln 
red, which gradually changes to purple, then to blue, and finally 
to bluish green. Color reaction is very distinct with 5 mg 
monocarboxylic acid, and amts as low as 1 mg can usually be 
detected. Therefore no pyrethrin I should be reported if color 
reaction is neg. 

With samples contg much perfume or other saponifiable 
ingredients, it may be necessary to use as much as 50 mL 1/V 
ale. NaOH. When lethanes are present, after washing HgCI ppt 
with alcohol and CHCI 3 , wash once more with alcohol and then 
several times with hot H 2 0. 



6.172 



Determination of Pyrethrin if {50) 



If necessary, filter aq. residue from pet ether extn thru gooch. 
Cone, filtrate to ca 50 mL and transfer to 500 mL g-s separator. 
Wash beaker with three 15 mL portions H 2 0. Acidify with 10 mL 
HCl and sat. with NaCI. {Acidified aq. layer must contain visible 
NaCI crystals thruout following extns.) 

Ext with 50 mL ether, drain aq. layer into second separator, 
and ext again with 50 mL ether. Continue extg and draining aq. 
layer, using 35 mL for third and fourth extns. Shake each ext 5=1 
min, releasing pressure, if necessary, by inverting separator and 
carefully venting thru stopcock. Let layers sep. 3=5 min or until 
aq. layer is clear before subsequent draining and extn. Combine 
ether exts, drain, and wash with three 10 mL portions satd NaCI 
soln. Filter ether exts thru cotton plug into 500 mL erlenmeyer 
and wash separator and cotton with addnl 10 mL ether. Evap. 
ether on H 2 bath, and remove any fumes of HCl with air current 
and continued heating ^5 min. Dry 10 min at 100°. 

(a) For crude pyrethrum exts. — Treat residue with 75 mL 
boiling H 2 and filter thru 9-11 cm Whatman No. 1, or equiv., 
paper. Wash flask and paper with five 20 mL portions boiling 
H 2 or until filtrate from final wash is neut. to litmus. Add 1-2 
drops phthln and rapidly titr. with 0.02/V NaOH (1 mL = 0.00374 
g pyrethrin H). Check normality of 0.02/V NaOH same day sample 
is titrd. 

(b) For refined pyrethrum exts. — Add 2 mL neut. alcohol and 
20 mL H 2 0, and heat to dissolve acid. Cool, filter thru gooch if 
necessary, add 1-2 drops phthln, and titr. with 0.02/V NaOH (1 
mL = 0.00374 g pyrethrin II). Check normality of 0.02/V NaOH 
same day as sample is titrd. 



Sabadilla Alkaloids {51) — Official Final Action 

(In dust formulations) 



6.173 



Determination 



[Caution: See 51.011, 51.040, and 51.056.) 

Weigh 10 g mixed 50% sabadilla dust (or corresponding amt 
of lesser concn) into 500 mL g-s erlenmeyer. Add exactly 300 
mL ether-CHCI 3 (3 + 1), and shake 5 min. Make alk. with 10 mL 
NH 4 OH and shake mech. 2 hr. Let stand overnight; then shake 
1 hr. 



Filter, avoiding evapn. Place 200 mL aliquot in 500 mL sepa- 
rator, acidify with H 2 S0 4 (3+97), and shake; withdraw small amt 
aq. layer and test with litmus paper, returning soln to separator. 
Add 50 mL of the dil. H 2 S0 4 and shake. Let sep. and transfer 
acid ext to second 500 mL separator. Add 50 mL pet ether to 
acid ext and shake. Let layers sep. and transfer acid ext to third 
separator. Repeat extn of soln in first separator with two 50 mL 
portions of the dil. H 2 S0 4 , using same 50 mL pet ether in second 
separator for washing. Collect acid exts in third separator. 

Make acid exts alk. to phthln with NH 4 OH. Ext with three 50 
mL portions CHCI 3 . Wash each CHCI 3 ext by shaking gently with 
same 100 mL portion H 2 in fourth separator. (If emulsion 
forms, add small amt anhyd. Na 2 S0 4 .) 

Filter each CHCI 3 ext thru cotton into weighed 250 mL flask. 
Evap. CHCI 3 on steam bath. Add few mL alcohol, and evap. 
again. Dry 1 hr at 100° and weigh sabadilla alkaloids. Calc. % 
total alkaloids. 



6.174 



Qualitative Test 



Add 1-2 mL H 2 S0 4 to few mg of residue, 6.173. Presence of 
sabadilla alkaloids is indicated by yellow that gradually becomes 
intensely red with greenish fluorescence. 



TOBACCO AND TOBACCO PRODUCTS 
Nicotine 

{Note: Nicotine is very toxic. Avoid contact with skin.) 

Silicotungstic Acid Method {52)— Official Final Action 

(Includes nornicotine) 

6.175 Reagent 

Silicotungstic acid soln. — Dissolve 120 g silicotungstic acid 
(4H 2 O.Si0 2 .12W0 3 .22H 2 or Si0 2 .12W0 3 .26H 2 0) in H 2 and dil. 
to 1 L. (Acid should be white or pale yellow crystals, free from 
green color; soln should be free from cloudiness and green 
color. Of the several silicotungstic acids, 4H 2 O.SiO 2 .10WO 3 .3H 2 O 
and4H 2 O.SiO 2 .12WO 3 .20H 2 Odo not give cryst. pptswith nicotine 
and should not be used.) 



6.176 



Determination 



Weigh sample contg preferably 0.1-1.0 g nicotine. If sample 
contains very little nicotine (ca 0.1%), do not increase amt to 
point where it interferes with distn. Wash with H 2 into 500 mL 
Kjeldahl flask, and if necessary add little paraffin to prevent 
frothing and few small pieces pumice to prevent bumping. Add 
10 g NaCI and 10 mL NaOH soln {30% by wt), and close flask 
with rubber stopper thru which passes stem of trap bulb and 
inlet tube for steam. Connect trap bulb to well-cooled condenser, 
lower end of which dips below surface of 10 mL HCl (1+4) in 
suitable receiving flask. Steam distil rapidly. When distn is well 
under way, heat flask to reduce vol. of liq. as far as practicable 
without bumping or excessive sepn of insol. matter. Distil until 
few mL distillate shows no cloud or opalescence when treated 
with drop silicotungstic acid soln and drop HCl (1+4). Confirm 
alky of residue in distn flask with phthln. 

Adjust distillate, which may total 1.0-1.5 L, to convenient 
exact vol. (soln may be coned on steam bath without loss of 
nicotine), mix well, and pass thru dry filter if not clear. Test 
distillate with Me orange to confirm acidity. Pipet aliquot contg 
ca 0.1 g nicotine into beaker. (If samples contain very small amts 
of nicotine, aliquot contg as little as 0.01 g nicotine may be 
used.) To each 100 mL liq., add 3 mL HCl (1+4) and 1 mL 
silicotungstic acid for each 0.01 g nicotine supposed to be 



AOAC Methods (1980) 



Aldrin, Dieldrin, Endrin 



91 



present. Stir thoroly and let stand overnight at room temp. 
Before filtering, stir ppt to see that it settles quickly and is in 
cryst. form. Filter on either ashless paper or gooch and wash 
with HCI (1+1000) at room temp. Continue washing for 2 or 3 
fillings of filter after no more opalescence appears when few 
mL fresh filtrate is tested with few drops nicotine distillate. With 
paper, transfer paper and ppt to weighed Pt crucible, dry 
carefully, and ignite until ail C is destroyed. Finally heat over 
Meker burner ^10 min. Wt residue x 0.1141 = wt nicotine in 
aliquot. With gooch, dry in oven 3 hr at 105° and weigh. Wt 
residue x 0.1012 = wt nicotine in aliquot. 



HALOGESSSATED PESTICIDES 

ABdrin, Dieldrin, and Endrin — Official Final Action 

it Total Chlorine by Sodium Biphenyl & 
Reduction Method [53) 

6.177 Principle 

Org. halogen compds are decomposed by Na biphenyl and 
liberated halide ion is titrd by Volhard method or potentidmet- 
rically after extn with H 2 from reaction medium. Applicable to 
detn of aldrin, dieldrin, or endrin in dusts, granules, wettable 
powders, emulsifiable cones, and solns in absence of other org. 
Cl-contg compds. More than trace amts of H 2 and appreciable 
amts of org. compds contg labile H cause excessive consumption 
of Na biphenyl. Interference of S is avoided, when present, by 
special treatment. 

6.178 Reagents 

(a) Dilute nitric acid.~S% by wt. Add 60 mL HN0 3 to 945 mL 
H 2 0. 

(b) Sodium biphenyl reagent — 30% w/w. {Caution: See 
51.034 and 51.038.) Place 300 mL dry toluene and 58 g Na in dry 
2 L 3-neck flask equipped with adjustable speed sealed stirrer, 
inlet for N, and reflux condenser. With stirrer off, and with slow 
stream of N passing thru flask, warm until refluxing begins and 
Na is entirely melted. Agitate vigorously until Na is finely 
dispersed; then cool to <10°. Remove reflux condenser and add 
1 .25 L anhyd. ethylene glycol dimethyl ether. Add 390 g biphenyl 
with moderate stirring and with slow stream of N passing thru 
flask. Reaction should begin within few mm, indicated by blue 
or green color which gradually darkens to black. Maintain temp, 
at <30° with oil bath or other cooling medium not involving 
hazard should flask contg Na break. Reaction should be complete 
in 1 hr. Reagent protected from moisture and air has useful life 
of 1-2 months at 25°. 

(Premixed reagent, packed in 15 mL vials, each enough for 1 
detn, is available from Southwestern Analytical Chemicals, Inc., 
PO Box 485, Austin, TX 78767.) 

(c) Toluene. —Nitration grade, Cl-free. 



6.179 



Preparation of Sample 



(Caution: See 51.041 and 51.084.) 



(a) Technical products. — Accurately weigh ca 0.1 g sample 
into 125 mL separator contg 25-30 mL toluene. Cautiously add 
10-14 g Na biphenyl reagent, mix by swirling, and let stand 5 
min. If soln is not dark green, add addnl 10-14 g reagent. 
(Dieldrin and endrin require 15 min reaction time after final addn 
of reagent.) 

Destroy excess reagent by dropwise addn of H 2 0, shaking 
frequently between addns, until green color is completely re- 
moved. Then slowly add 25 mL dil. HN0 3 , with intermittent 
swirling. Stopper separator, and mix with gentle rocking motion, 



venting occasionally. Avoid vigorous shaking during this first 
extn. 

Let sep., rinse stopper and walls of separator with H 2 0, and 
drain aq. phase into 250 mL g-s erlenmeyer. Re-ext reaction 
mixt. with two 25 mL portions dil. HN0 3 , shaking vigorously. 
Add aq. exts to erlenmeyer and det. CI. 

(b) Emulsifiable concentrates and oil spray solns.— Mix tho- 
roly and weigh sample contg 0.05-0.08 g CI into 250 mL 
separator contg 25-30 mL toluene. Proceed as in (a), beginning 
"Cautiously add . . ." 

(c) Dusts, granules, and wettable powders. — Weigh sample 
contg 0.1-0.15 g active ingredient in paper Soxhlet extn thimble, 
place in extn app., and ext with ca 150 mL acetone in 300 mL 
flask 3 hr. Evap. ext to dryness on steam bath, dissolve residue 
in few mL toluene, and quant, transfer to 250 mL separator, 
using 25-30 mL toluene. Continue as in (a), beginning ''Cau- 
tiously add ..." 

If S is brought into soln by decomposition of emulsifiers or 
other compds such as org. thiophosphates, remove as follows: 
Add 30% NaOH soln to acid soln in erlenmeyer until alk. to 
phthln, and add 1 mL excess. Add 5 mL 30% H 2 2 , heat to bp 
on hot plate, and boil ca 10 min. Let cool slightly, cautiously add 
5 mL more 30% H 2 2 , and boil again ca 10 min. Cool, and add 
small flake (ca 0.05 g) hydrazine sulfate to remove last traces of 
H 2 2 . Neutze with dit. HN0 3 to phthln and add 2-3 mL excess. 



6.180 



Determination 



(a) Colorless solns.— To acid aq. soln add 30 mL H 2 0, 10 mL 
nitrobenzene, 3 mL ferric indicator, 6.019(e), and, from buret, 
0.4-0.6 mL 0.05/V KSCN. Swirling constantly, titr. with 0.1/V 
AgN0 3 until red is discharged, and add 2-5 mL excess. Stopper 
flask tightly and shake vigorously 15 sec. Without refilling buret, 
titr. slowly with the 0.05/V KSCN until end point approaches. 
Stopper flask, shake vigorously 20-30 sec, and continue titrn 
until 1 drop produces distinct reddish color which does not fade 
on swirling or vigorous shaking. 

(b) Colored solns or chloride in presence of bromide and /or 
iodide. — To acid aq. soln add 30 mL H 2 0, transfer to 400 mL 
beaker, adjust vol. to 200-250 mL, and add 0.5 g Ba{N0 3 ) 2 . Titr. 
with 0.1/V AgN0 3 potentiometrically, with stirring, using cell 
system of either glass ref. electrode and Ag indicating electrode 
or Ag-AgCI electrode system, electronic voltmeter, and 10 mL 
buret. 

(c) Blank determination. — Det. blank on all reagents by adding 
10-14 g Na biphenyl reagent to 25 mL toluene and continuing 
as in 6.179(a). 



6.181 



Calculations 



% Aldrin, dieldrin, or endrin = (net mL AgN0 3 - mL blank) 
x normality x 35.45 x /7(10 x g sample), where F is 1.61 for 
aldrin, 1.81 for dieldrin, and 1.74 for endrin. Net mL AgN0 3 = 
[mL 0.1/V AgN0 3 - (mL 0.05/V KSCN/2)]. 

(For most accurate results, det. factor F for specific batch of 
tech. pesticide used in formulation. Toxicant content is stenciled 
on drum. Calc. F - PjC, where P - % purity (toxicant content 
as stenciled on drum), and C = % CI by wt.) 



infrared Method (53) 



6.182 



Principle 



Dieldrin and endrin in dusts, granules, wettable powders, 
emulsifiable cones, and solns are purified on adsorbent columns. 
Hexachloro-epoxy-octahydro-endo,exo-dimethanonaphthalene 

* Surplus method— see inside front cover. 



92 



6. Pesticide Formulations 



AOAC Methods (1980) 



(HEOD) content of the purified dieldrin or of tech. dieldrin is 
detd by IR, using baseline technic, and dieldrin is calcd assuming 
85% HEOD content. Endrin content of purified or tech. endrin 
is detd as hexachloro-epoxy-octa-hydro-endo,endo-dimethan- 
onaphthalene similarly. 

Aldrin is extd from dusts, wettable powders, and inorg. 
fertilizers on adsorbent column. Hexachloro-hexahydro- 
endo,exo-dimethanonaphthalene (HHDN) content of the ext or 
of tech. aldrin is detd by IR, using baseline technic, and aldrin 
is calcd assuming 95% HHDN content. Method is not applicable 
to emulsifiable cones or granules contg petroleum hydrocarbon 
solvs or to mixts contg other common pesticides or adjuvants 
that absorb in same wavelength region as HHDN. 



6.183 



Reagents and Apparatus 

Mix 1 vol. CHCU with 19 



(a) Chromatographic solvent A 
vols hexane. 

(b) Chromatographic solvent B. — Mix 1.5 vols acetone with 
98.5 vols chromtgc solvent A. 

(c) Extraction solvent — Mix 1 vol. acetone with 19 vols CS 2 . 

(d) Infrared spectrophotometer, — With sealed liq. cells with 
NaCI windows, having optical path length of ca 0.1 mm (dieldrin 
and endrin) and 0.2 mm (aldrin). 



6.184 



Preparation of Standard Solutions 



(a) HEOD std soln for dieldrin. — Accurately weigh ca 100, 200, 
300, 400, 500, and 600 mg std hexachloro-epoxy-octahydro- 
endo,exo-dimethanonaphthalene (HEOD) into 10 mL vol. flasks, 
dissolve in CS 2 , and dil. to vol. Concns will be 1, 2, 3, 4, 5, and 
6 g/100 ml_, resp. 

(b) Std soln for endrin. — Accurately weigh ca 50, 100, 150, 
200, 300, and 400 mg std hexachloro-epoxy-octahydro- 
endo,endo-dimethanonaphthalene (endrin) into 10 mL vol. 
flasks, dissolve in CS 2 , and dil. to vol. Concns will be 0.5, 1.0, 
1.5, 2.0, 3.0, and 4.0 g/100 mL, resp. 

(c) HHDN std soln for aldrin. — Accurately weigh ca 100, 150, 
200, 250, 300, and 350 mg std hexachloro-hexahydro-endo,exo- 
dimethanonaphthalene (HHDN) into 10 mL vol. flasks, dissolve 
in CS 2 , and dil. to vol. Concns will be 1.0, 1.5, 2.0, 2.5, 3.0, and 
3.5 g/100 mL, resp. 

6.185 Preparation of Standard Curve 

Fill 0.1 mm cell (0.2 mm for aldrin) with most dil. of stds solns, 
using hypodermic syringe. Adjust spectrophtr to optimum set- 
tings for gain, slit width, response, speed, and drum drive. Make 
duplicate scans of CS 2 soln over scanning range indicated in 
Table 6:01 and repeat with each of other std solns at same 
instrument settings. 

For each of scans of the 6 std solns of each compd, draw line 
between baseline points indicated in table. Draw perpendicular 
from zero radiation line thru absorption peak to baseline and 
measure distance from line to peak, P, and to baseline P . 
Calc. A (= log PJP) and plot as ordinate against concn in g/100 
mL as abscissa. 

Since std curve intersects abscissa at pos. concn value, method 
is not applicable to concns below this value. 

Peak wavelengths given in table are characteristic for low 

Table 6:01 Characteristic Wavelength Points for infrared 
Determination of Dieldrin, Endrin, and Aldrin, jitm 



Compound 



Scanning 
Range 



Baseline 
Points 



Peak at 
Low Concn 



HEOD 

Endrin 
HHDN 



11.59-12.18 
11.43-12.04 
11.79-12.24 



11.64, 12.18 
11.50, 11.97 
11.85, 12.24 



11.80 
11.76 
12.01 



concns and they shift at higher concns. P is always detd as 
distance from line to point of max. absorption. 

6.186 Preparation of Sample 

[Caution: See 51.011, 51.041, 51.046, 51.056, and 51.061.) 

(a) Dusts and wettable powders. — Transfer 3-20 g sample, 
depending on concn (75-0.5%), weighed to nearest 0.01 g, to 
chromatgc tube contg 25—50 mm (ca 5.5 g) Hyflo Super-Cel. 
(For finely divided dieldrin or endrin powder, use 3 g activated 
C instead of Super-Cel.) Tamp or vibrate column slightly to 
settle contents. Place 250 mL wide-mouth erlenmeyer or 500 
mL evapg dish under tip of column. 

Working in well ventilated hood, add 50 mL portions extn 
solv. to column (if S is present, ext with acetone instead of extn 
solv.), letting solv. percolate thru column between addns, until 
150 mL ext collects. Rinse tip of column with addnl 10 mL extn 
solv. 

Evap. solv. almost to dryness on steam bath under N. Dry 
HEOD or HHDN residues 15 min at 75°; dry endrin in vac. oven 
15 min at 30° and 10 mm pressure. (Extd endrin may no longer 
be associated with its inhibitors. Residue must not be exposed 
to elevated temps and must be dissolved promptly to avoid 
decomposition.) 

Cool residue and dissolve in few mL CS 2 . Quant, transfer to 
vol. flask of such size (5-100 mL) as to give optimum concn of 
3 g HEOD, 2 g endrin, or 2 g HHDN/100 mL, dil. to vol. with CS 2 , 
and mix thoroly. If soln is cloudy from H 2 0, add little NaCI, 
shake, and let settle. 

(b) Granules containing dieldrin or endrin. — Slurry 40 g Flo- 
risil in 200 mL beaker with 1 00 mL hexane. Transfer to chromatgc 
column with stream of hexane from wash bottle. Eliminate any 
bubbles or voids by vibration or agitation. Let hexane drain until 
only 2-3 mm layer remains above surface of column. Add small 
layer of Na 2 S0 4 to top of column. 

Transfer 2-10 g finely ground sample, depending on concn 
(10-1%), to prepd column. Rinse down column walls with three 
10 mL portions chromatgc solv. A, letting each portion enter 
column before adding next. Add 170 mL chromatgc solv. A, let 
percolate thru column, and discard. 

Gently flow 10 mL chromatgc solv. B down walls of tube, 
avoiding disturbing surface of adsorbent. After solv. sinks into 
column, repeat washing with 2 addnl 10 mL portions. Add 220 
mL chromatgc solv. B and let flow at rate of 2-5 mL/min, 
collecting eluate in 500 mL wide-mouth erlenmeyer or evapg 
dish. Evap. solv. to dryness on steam bath, avoiding spattering, 
and proceed as in (a), using 5-10 mL vol. flask. 

(c) Emulsifiable concentrates and so/ns.— Weigh 1.5 g dieldrin 
cone. (1.5 lb/gal.), 1.0 g endrin cone. (1.6 lb/gal.), or 30.0 g 0.5% 
dieldrin soln, and add 5 mL hexane. Transfer to prepd column 
and proceed as in (b). 

(d) Technical materials, — Transfer sample contg 1.75-4.00 g 
dieldrin, 1.50-3.00 g endrin, or 1.00-2.00 g aldrin, weighed to 
0.01 g, to 100 mL vol. flask. Dissolve in CS 2 and dil. to vol. with 
CS 2 . 



6.187 



Determination 



Fill same 0.1 mm cell (0.2 mm for aldrin) used for prepn of std 
curve with sample soln. Make duplicate scans, and calc. A and 
mean A as in prepn of std curve. From appropriate std curve, 
obtain g HEOD, endrin, or HHDN/100 mL sample soln, W. 

% dieldrin = W x V x 1.175/S; 

% endrin = W x V/S; 

% aldrin - W x V x 1.053/S; 
where V = mL sample soln; S = g sample; 1.175 and 1.053 = 
conversion factors HEOD to dieldrin and HHDN to aldrin, resp. 



AOAC Methods (1980) 



BHC 



93 



Amiben (3-Amino-2,5-dichlorobenzoic 
Acid) (54)— Official Final Action 

AOACCIPAC Method 

6.188 Principle 

Amiben contains conjugated n electron system of benzene 
which absorbs strongly in UV. Absorption is measured quant, 
at 297 nm. {Caution: See 51.018 and 51.041.) 

6. 1 89 Apparatus and Reagents 

(a) Spectrophotometer. — For use in UV, with 1 cm cells. 

(b) Shake-out flask. — 250 mL erlenmeyer, with screw cap. 

(c) Amiben std solns. — (1) Stock soln. — 0.38 mg/mL. Accu- 
rately weigh 19±2 mg amiben (Amchem Products, Inc., Ambler, 
PA 19002) into 50 mL vol. flask, add 25 mL 1% NaOH, agitate 
until dissolved, dil. to vol., and mix. (2) Working soln. — 0.038 
mg/mL. Pipet 5 mL stock soln into 50 mL vol. flask, dil. to vol. 
with 1% NaOH soln, and mix. 

6. 1 90 Preparation of Sample 

Mix 10 g granular sample on 12 x 12" paper by lifting alternate 
corners. 



6.191 



Determination 



(a) Dry granular formulations. — Add amt solid material and 
1 % NaOH soln specified in Table 6:02 to 250 mL shake-out flask 
and shake 30 min. Filter, and transfer stated aliquot to vol. flask. 
Dil. to vol. with 1% NaOH soln and mix. Det. A at 360 and 297 
nm against 1% NaOH. Calc. A A = A 2Q7 - A 360 . Det. A/4' of 
working std soln similarly. 

% Amiben = {AA x (mgstd/mL) xF)/{AA' x g sample x 10), 
where F = factor in Table 6:02. 

(b) Liquid formulations.— Weigh amt liq. indicated in Table 
6:02 into 100 mL vol. flask, dil. to vol. with 1% NaOH soln, and 
mix. Transfer 1 mL aliquot to 100 mL vol. flask, dil. to vol. with 
1% NaOH, and mix. Proceed as in (a). 



Benzene Hexachloride (BHC) (Hexachlorocyclohexane) 
Gamma Isomer (Lindane) 

Partition Chromatographic Method (55) 
Official Final Action 

{Caution: See 51.009, 51.01 1, 51.039, 51.040, 51.041, and 51.054.) 

6.192 Apparatus 

(a) Partition column. — Column and type reduction valve are 
shown in Fig. 6:04. Construct column of heavy-wall Pyrex tubing 
ca 3.5 mm thick, 90 cm long x 2.5 cm diam. Seal coarse porosity 
fritted glass disk in place and attach No. 18/9 f joint 5 cm below 
disk. Supply pressure from laboratory supply line. (Column 
available from SGA Scientific, Inc.; specify Cat. No. JC 1800 
constructed from heavy rather than std wall tubing.) 

(b) Solvent evaporator.— Fig. 6:04. Evap. fractions to dryness 



under reduced pressure at 60°, with aid of H 2 pump. Recover 
solv. in trap consisting of Kjeldahl flask immersed in mixt. of 
NaCl and ice. 

(c) Melting point apparatus. — Use Thiele mp a pp. equipped 
with mech. stirrer. App. shown in Fig. 6:05, or Hershberg 
modification {56) (available from Ace Glass, Inc., Cat. No. 7686) 
is suitable. 

(d) Thermometer. — Precision grade, meeting NBS specifica- 
tions: partial immersion; range 90-120° in 0.2° subdivisions. 
Calibrated by NBS or against thermometer checked by NBS. 

(e) Melting point tubes. — 1-2 mm capillary tubes of uniform 
wall thickness and diam. 



6.193 



Reagents 



(a) n-Hexane. — Com. grade, distd before use. 

(b) Nitromethane.— Hei\ux com. grade material 4 hr and distil. 
No visible residue is left after evapn of 10 mL purified material. 

(c) Silicic acid. — Use Mallinckrodt reagent grade (for chro- 
matgy) which meets following requirements: When column 
prepd as in 6.195 is used for detn on sample contg known amt 
of y-isomer, flow rate and packing characteristics should be 
similar to those of an H 2 Si0 3 known to be satisfactory, and 
recovery of y-BHC should be within ±3% of the y-BHC content. 

(d) Dye soln.— Dissolve 25 mg D&C Violet No. 2 (1-hydroxy- 
4-p-toluidino-anthraquinone) in 50 mL mobile solv. and store in 
g-s bottle. (Available from Aldrich Chemical Co.) 

(e) Mobile solvent — Satd soln nitromethane in /?-hexane. 
Vigorously shake 2 L A?-hexane with excess nitromethane in 
g-s bottle. Decant mobile solvent from nitromethane as needed. 



6.194 



Preparation of Sample 



(a) Powders containing more than 10% y-BHC. — Crush and 
thoroly mix sample with mortar and pestle. Weigh enough 
sample into tared 125 mL erlenmeyer to provide ca 0.2 g y- 
isomer after extg and aliquoting. Add 25 mL mobile solv., heat 
just to bp on steam bath, and cool to room temp., shaking 
occasionally. Decant ext thru buchner with ca 34 mm medium 
porosity fritted disk into 100 mL Kohlrausch flask, with gentle 
suction. Re-ext residue in flask, using 10 mL mobile solv. Wash 
residue and flask with five 10 mL portions cold mobile solv., 
decanting each wash thru buchner. Add 2 mL dye soln and dil. 
to vol. with mobile solv. 

(b) Dusts containing less than 10% y-BHC. — Weigh enough 
sample to provide 1.75-2.00 g y-isomer. Transfer to Soxhlet 
extractor and ext overnight with ether. Evap. most of ether on 
steam bath and evap. remainder at room temp, under vac. Ext 
y-isomer from residue with mobile solv. as in (a). 

6.195 Preparation of Column 

{Caution: See 51.004 and 51.061.) 

Transfer 100±0.5g H 2 Si0 3 to high-speed blender, add 300 mL 
mobile solv., and with mixing, add 55 mL nitromethane. Mix 
15-30 sec; then pour into column thru glass funnel. Stir slurry 
with long glass stirring rod to displace air bubbles. Wash down 





Table 6:02 Parameters for sample 


analysis 






Sample 


Amiben, 
% 


Sample 

Wt, 
g±0.1 


1% NaOH, 
mL 


Aliquot, 
mL 


Final 
Diln 


Factor 
{F) 


Dry granular 
Dry granular 
Dry granular 
Liquid 


1.2 

4 
10 
21.6 


3.0 
7.5 
3.0 
1.8 


50 

100 
100 
100 


2 
1 
1 
1 


50 
100 
100 
100 


1,250 
10,000 
10,000 
10,000 



94 



6. Pesticide Formulations 



AOAC Methods (1980) 





FIG. 6:04 — Partition column and solvent evaporator 



sides of column with few mL mobile solv. and apply 5 lb 
pressure to pack column and force out excess solv.; tap column 
gently to aid packing. When boundary between solv. and H 2 Si0 3 
remains stationary, release pressure cautiously, pipet out most 
of excess solv., and reapply pressure until ca3 mm solv. remains 
above adsorbent. 



6.196 



Determination 




FIG. 6:05— Melting point apparatus 



{Caution: See 51.015 and 51.018.) 

Pipet 10 mL aliquot of sample soln onto column by letting it 
flow slowly down inside of column without disturbing H 2 Si0 3 
surface. Wash down side of column with 2 mL mobile solv. and 
force soln into column by applying 2-3 lb pressure, releasing 
pressure when all solv. has entered column. Add 10 mL mobile 
solv. and force into column. Release pressure and slowly add 
mobile solv. to within 7-12 cm from top of column. Apply 
enough pressure to force solv. thru column at 3-4 mL/min. Just 
before last trace of dye leaves column, begin to collect 10 mL 
fractions, alternately using two 10 mL graduates. Transfer each 
fraction to 125 mL erlenmeyer and evap. to dryness, using solv. 
evaporator. (Evap. fractions without boiling; if boiling begins, 
raise flask momentarily from H 2 bath.) 

Appearance of y-isomer upon evapn is recognized by its 
tendency to cover bottom of flask as white residual film with 
typical crystal formation. When first residue of y-isomer is 
recognized, begin to collect 10 mL fractions until all y-isomer is 
obtained (usually ^8 fractions). Dissolve residue in each flask 
with 5 mL /?-hexane and transfer to weighed flask, rinsing flasks 
successively with 5 mL portions n-hexane. Evap. solv., using 
solv. evaporator. Evacuate flask ca 20 min at room temp, with 
vac. pump. (There is little danger in evacuating 125 mL erlen- 
meyer; larger size erlenmeyer, however, is likely to collapse 
under vac.) Release vac, wipe with clean, moist towel, and let 
stand 5 min. Weigh, and calc. % y-benzene hexachloride in 
original sample. 

6.197 Melting Point Determination 

of the Gamma Fraction 

Dissolve residue in min. amt acetone and transfer quant, to 
10 mL beaker. Evap. acetone at 40°, using filtered air stream. 
Scrape residue from beaker for mp detn, (Beaker may be set on 
piece of solid C0 2 to ensure prepn of finely powd product.) Place 
material in agate mortar and mix thoroly with pestle. 

Select 2 clean, dry capillary tubes and fill with sample. Be 
sure material is well packed into bottom of tube to ensure max. 
contact between sample and wall of tube. Insert tubes and 



AOAC Methods (1980) 



Balan or Trifluralin 



95 



thermometer bulb in Thiete tube so that samples and thermom- 
eter bulb touch. Start stirrer and heater, and adjust heating rate 
to r/min at 90°. Continue heating until sample melts or reaches 
106°. Reduce heating rate to 0.5°/min and continue heating until 
sample melts. 

Sample mp is corrected temp, of bath when last solid disap- 
pears into the clear melt If mp is <108°, check result by IR 
method, 6.198-6.201. 

Infrared Spectrophotometry Method [57) 
Official Final Action 

(Applicable to tech. BHC. Caution: See 51.018, 
51.041, and 51.048.) 



6.198 



Apparatus 



Infrared spectrometer. — With matched pair of Nq. absorption 
cells. 0.5-1.1 mm thick. 



6.199 



Calibration of Cells 



Det, in spectrometer, difference between deflections of the 
2 cells filled with CS 2 . Plainly mark one cell to be used as sample 
cell for reading /. Correct values of / obtained with other cell by 
adding or subtracting difference between cells and refer to this 
as cell factor F, Check factor every 10-14 days. 



6.200 



Preparation of Standards and Working Curves 



Obtain a, fi, y, and 5 isomers of BHC, either by fractional crystn 
from tech. material or as sepd materials, and recrystallize several 
times from solvs that have been redistd from all-glass app. 
Recrystallize from following solvs until mps by capillary tube 
method become const: a isomer from benzene followed by 
MeOH (mp ca 158°); fi isomer from toluene (mpca 210.5°, sealed 
capillary); y isomer from MeOH (mp ca 113°); and 8 isomer from 
CCI 4 followed by CHCI 3 (mp ca 138.5°). 

Confirm purity of each isomer as follows: Evap. to dryness 
enough mother liquor from last crystn to yield ^1 g dissolved 
solids, grind residue, and dry overnight in evacuated desiccator. 
Weigh and dissolve in enough CS 2 to make 4 g/100 mL soln. 
Prep, corresponding soln of recrystd isomer as std. Compare 
solns of residue and std in spectrometer at wavelength points 
used for analysis of other isomers. Consider purity of isomer 
satisfactory if A of residue soln is not significantly greater than 
that of std at these points. 

Prep, working curves of the isomers by detg T of their solns 
in CS 2 at various conens as in 6.201. Calc. A and plot against 
concn in g/L. 



6.201 



Determination 



Reduce sample of tech. BHC to ca 2 g by grinding and 
quartering, and dry 24 hrin vacuo at room temp. Weigh 1.5000 
g dried material into 50 mL vol. flask and dit. to vol. with CS 2 
(equiv. to 30 g/L). Shake vigorously to dissolve {(3 isomer is not 
completely sol. and will settle out). Pipet 25 mL of this sample 
soln into another 50 mL vol. flask and again dil. to vol. with CS 2 
(equiv. to 15 g/L). Fill sample cell with the coned soln for reading 
/, and fill blank cell with CS 2 , place in spectrometer, and read T 
in duplicate at following wavelengths: 





Wavelength, fxm 


Alpha 


12.58 


Beta 


13.46 


Gamma 


14.53 


Delta 


13.22 


Epsilon 


13.96 



Average duplicates for calcns. Repeat readings with dil. soln 
(15 g/L) at a and y wavelengths. Calc. A of each of isomers at 
the various wavelengths from T measurements by equation: 

. {Fxl b )-(Fxl b x%Sct) _ 
9 / s - {F x /„ x % Set) 
where F - cell factor, / b = reading of blank cell, % Set - % 
scatter, / s = reading of sample cell, and>4 = absorbance. 

Obtain approx. conens from working curves, 6.200. Correct >4 
at each wavelength for absorption of interfering components. 
(Altho /3 isomer has low solubility in CS 2 , this isomer interferes 
with 8 analytical point; therefore det. ,4 of p isomer in CS 2 at this 
point and apply as correction.) Since these new values are 
overcorrected, make repeated evaluations until successive val- 
ues are const, within desired precision. 

6.202 * Radioactive Tracer Method [58) * 

Official First Action 

See 6.257-6.260, 11th ed. 

/V-ButyB-A(-ethyS-a,a,a-frifluoro-2 r 6-dinitro-p-toluidine 

(Balan®) or Trifluralin {a,a,a-Trifluoro-2,6-dinitro-/V,/V- 

dipropyl-p-toluidine [59) — Official Final Action 

Ultraviolet Method 

6.203 Principle 

Trifluralin or Balan is extd from solid carrier or dissolved in 
n-hexane if liq., purified by chromatgy on Florisil, and detd by 
UV spectrometry at 376 nm. 



6.204 



Reagents 



(a) Florisil. — 100-200 mesh. Test elution characteristics of 
Florisil by adding 5 mL std soln to prepd column. Proceed as in 
6.207. Elution vol. should be s*80 mL but <100 mL If elution 
vol. does not fall within this range, adjust H 2 content of Florisil 
by trial and error to obtain proper elution (add H 2 to decrease 
elution time; dry at 130° to increase it). 

(b) Std so//i. —1.25 mg/mL. Weigh 0.125 g trifluralin or Balan 
Ref. Std (Elanco Products Co.), into 100 mL vol. flask, dil. to vol. 
with /7-hexane, and mix. 



6.205 



Preparation of Column 



Insert glass wool plug in bottom of 25 x 400 mm glass tube 
with Teflon stopcock. Add, with const tapping of column, 5 g 
anhyd. Na 2 S0 4 , stdzd Florisil, (a), to ht of 50 mm, and 5 g anhyd. 
Na 2 S0 4 . With stopcock open, add 50 mL /7-hexane and let drain 
to top of column. Close stopcock. 

6.206 Preparation of Sample 

(a) Dry formulations {containing more than 7% trifluralin or 
Balan). — Weigh sample contg 0.25 g trifluralin or Balan into 
Soxhlet extn thimble (33 x 80 mm), cover with glass wool, and 
ext with CHCI 3 1 hr beyond time when no further color is extd. 
Quant, transfer ext to 200 mL vol. flask with CHCI 3 , dil. to vol. 
with CHCI3, and mix. Transfer 5 mL to r-b flask and evap. just 
to dryness on rotary evaporator. 

(b) Dry formulations {containing 1% or less trifluralin or 
Balan).— Weigh sample contg 0.05 g trifluralin or Balan, ext, 
transfer to 200 mL vol. flask, and dil. as in (a). Transfer 25 mL 
to r-b flask and evap. just to dryness on rotary evaporator. 

(c) Liquid formulations. — Weigh sample contg 0.12 g triflur- 
alin or Balan into 100 mL vol. flask. Dil. to vol. with n-hexane 
and mix vigorously. Proceed as in 6.207. 

* Surplus method— -see inside front cover. 



96 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.207 



Determination 



Transfer 5 mL soln from (c) or residue from (a) or (b), with aid 
of n-hexane, to Florisil column. Transfer 5 mL std soln to second 
Florisil column. Wash sample into column with small portions 
/7-hexane. Let each portion drain to top of column before adding 
next. Fill column with /7-hexane, discarding eluate until band 
has moved ca % length of column. Collect eluate contg trifluralin 
or Balan band (first yellow-orange band to elute) in 100 mL vol. 
flask. (If band requires >100 mL vol. to elute, replace vol. flask 
with r-b flask, evap., and transfer quant, to 100 mL vol. flask.) 
[Caution: See 51.011(a) and 51.061.) Dil. to vol. with n-hexane 
and mix. Det. A of sample and std solns in 1 cm cells at 376 nm 
against n-hexane as ref. 

6.208 Calculations 

% Trifluralin or Balan = {A x g std x F x P)/{A' x g sample), 
where A and A' refer to sample and std solns, resp.; P = % 
purity of std; and F - 2, 0.4, or 1 for sample prepns (a), (b), or 
(c), resp. 

Gas-Liquid Chromatographic Method 

6.209 Principle 

Trifluralin or Balan is extd from solid carrier, or dissolved in 
acetone if liq., and detd by GLC. 

6.210 Reagents 

(a) Diisobutyi phthalate internal std soln. — Weigh 0.625 g 
diisobutyl phthalate (ICN-K&K Laboratories, Inc.) into 250 mL 
vol. flask, dil. to vol. with acetone, and mix. 

(b) Std so/a— -1.6 mg/mL Weigh 0.16 g trifluralin or Balan 
Ref. Std into 100 mL vol. flask, dil. to vol. with acetone, and mix. 



6.211 



Apparatus 



(a) Gas chromatograph. — Equipped with flame ionization de- 
tector; capable of programmed column temp, from 135 to 190° 
at 8°/min. Approx. instrumental conditions: inlet 205°, detector 
275°, N carrier gas 60 mL/min. 

(b) Column. — 1.5 m (5') x % or %" od, stainless steel or 
Pyrex glass tube packed with 5% DC 200, 12,500 cstokes 
(Analabs, Inc.) on 80-100 mesh Chromosorb W (HP). Condition 
newly prepd column at 230° overnight with N carrier gas. 

6.212 Preparation of Sample 

(a) Dry formulations {containing more than 1% trifluralin or 
Balan). — Weigh sample contg 0.16 g trifluralin or Balan into 
Soxhlet extn thimble {33 x 80 mm), cover with glass wool, and 
ext with acetone 1 hr beyond time when no further color is extd. 
{Caution: See 51.011(a) and 51.046.) Evap. to ca 60 mL on steam 
bath with stream of air directed into flask. Transfer quant, to 
100 mL vol. flask with acetone. Dil. to vol. with acetone and mix. 

(b) Dry formulations {containing 7% or less trifluralin or 
Balan). — Weigh sample contg 0.04 g trifluralin or Balan, ext, and 
evap. as in (a). Transfer quant, to 100 mL vol. flask with acetone 
and proceed as in 6.213 without dilg, beginning, ". . . add 10 mL 
internal std soln, . . ." 

(c) Liquid formulations. — Weigh sample contg 0.16 g triflur- 
alin or Balan into 100 mL vol. flask, dil. to vol. with acetone, and 
mix. 



6.213 



Determination 



Pipet 25 mL acetone soln, 6.212(a) or (c), and 25 mL std soln, 
(b), into sep. 100 mL vol. flasks, add 10 mL internal std soln, dil. 
to vol. with acetone, and mix. 



Inject 2.5 ju.L trifluralin or Balan std soln and start temp, 
program to give symmetrical peak ca 70% scale deflection and 
retention time 5.5 min. Diisobutyl phthalate internal std peak 
appears ca 2 min after std peak. Repeat injection of std soln 
until ratio of trifluralin or Balan peak area to internal std peak 
area is reproducible. 

Without changing conditions inject 2.5 ^tL sample soln. 

6.214 Calculations 

Calc. areas of trifluralin or Balan and diisobutyl phthalate 
peaks. Divide area of trifluralin or Balan peak by area of diisobutyl 
phthalate internal std peak to det. ratio, R. 

% Trifluralin or Balan = (/? x W x P)/{R' x W x f ), 
where R and R' = ratio for sample and std solns, resp.; W and 
W = g sample and std, resp.; P = % purity of std; and F = 1, 
1, or 4 for sample prepns (a), (c), or (b), resp. 

Captan (N-(Trichloromethylthio)-4-cyclohexene- 
1,2-dicarboximide) (G0)~-Official First Action 

AOACCIPAC Method 



{Caution: See 51.039 and 51.041.) 



6.215 



Principle 



Captan is extd from inerts with soln contg dieldrin in dioxane. 
Ratio of captan peak ht to dieldrin peak ht in gas chromatgy is 
measured and compared to ratiofrom std captan prepd similarly. 
Method applies to tech. and dry formulated products contg 
captan as only active ingredient. 

6.216 Reagents 

(a) Extracting soln.— Weigh ca 5.0 g dieldrin (ca 85% HEOD) 
into 500 mL vol. flask, add ca 300 mL 1,4-dioxane, shake to 
dissolve dieldrin, and dil. to vol. with dioxane. 

(b) Captan std soln. — Accurately weigh, by difference, 0.25 g 
captan ref. std into glass vial. Pipet 25 mL extg soln into vial, 
stopper, and shake until dissolved. Prep, fresh after 24 hr. 



6.217 



Gas Chromatography 



Use any app. that will completely sep. captan from HEOD and 
with following conditions: Sample inlet port 10-30° higher than 
column; thermal conductivity or H flame detector maintained 
5=20° higher than column (typical column temp. 220°); 1.5 m (5') 
x %" od glass column packed with 3% XE-60 silicone nitrile 
gum rubber on Chromosorb G, acid-washed and dimethyl 
dichlorosilane-treated {allowable variations are 0.6-1 .8 m (2-6'), 
%-%" od, 2-10% liq. loading, Chromosorb W). Condition newly 
prepd column at 230° overnight while purging with carrier gas 
(He for thermal conductivity or N for H flame detectors). 

6.218 Preparation of Sample 

Accurately weigh, by difference, well mixed portion of sample 
contg ca 0.25 g captan into 30 mL glass vial with plastic-lined 
screw cap. Into vial pipet 25 mL same extg soln used for prepn 
of std soln. Stopper and shake mech. 15 min. Centrf. if necessary 
to ppt inerts. Sample supernate with syringe for captan detn. 
Prep, fresh after 24 hr. 



6.219 



Determination 



Adjust column temp., carrier gas flow, injection size, and 
recorder attenuation so that captan and HEOD peaks are com- 
pletely sepd in <10 min and so that ht of HEOD peak, which 
elutes first, is ca % full scale. Repeat injections of std soln until 
peak ht ratio captan:HEOD of 2 consecutive injections varies 



AOAC Methods (1980) 



Chlordane 



97 



<2%. Without changing conditions, inject supernate from sam- 
ple. If sample ratio differs by >±10% from std ratio, reweigh 
sample. For detn, inject std, sample in duplicate, and std. 

Measure HEOD and captan peak hts. When solv. peak tailing 
extends into region of HEOD and captan peaks, draw curved 
extension as baseline. Det. peak ht ratio captan:HEOD and 
average the 2 values (/? s ) for std injections just before and after 
sample injections. Calc. and average peak ht ratio of the 2 
samples (/? x ). 

% Captan = (/? X /W X ) x (W s x P/Rs), 
where W s = g std, W x = g sample, and P = % purity of captan 
std. 



Technical Chlordane 
Total Chlorine Method [61) — Official Final Action 

{Caution: See 51.011, 51.018, 51.038, 51.039, 51.040, 51.041, 
and 51.045.) 



6.220 



Standardization of Standard Solutions 



(a) Sodium chloride std soln. — 0.1 N. Dissolve 5.845 g NaCI, 
previously dried 2 hr at 105°, in H 2 0, and dil. to 1 L in vol. flask. 

(b) Silver nitrate std soln.—OAN. Prep, as in 50.027. To 250 
mL g-s erlenmeyer add 15.00 rhL 0.1/V NaCI, (a), 50 mL H 2 0, 10 
mL HN0 3 (1+1), boiled to expel oxides of N, and 25.00 mL of 
the AgN0 3 soln. Add 3 mL nitrobenzene, stopper, and shake 
vigorously 15 sec. Add 5 mL ferric indicator, 6.019(e), and back- 
titr. with 0.1 N KSCN, (c), to reddish-brown end point. (Potenti- 
ometric titrn using Ag indicator electrode and Ag-AgCI or glass 
ref. electrode may be substituted for indicator method, but must 
be used in both stdzn and detn.) 

(c) Potassium thiocyanate std soln. — 0.1 N. Prep, and titr. 
against AgN0 3 soln, (b), as in 50.030(b). Calc. F = mL AgN0 3 
soln/mL KSCN soln. 

Normality AgN0 3 soln = mL NaCI soln x 0.1000/(mL AgN0 3 
soln - mL KSCN soln x F). 



6.221 

(a) Emulsifiable 



Preparation of Sample 



concentrate formulations. — Ace u rate ly 
weigh sample contg 0.5±0.05 g tech. chlordane into 50 mL vol. 
flask, dissolve, and dil. to vol. with toluene. Transfer 5 mL aliquot 
to 125 mL separator, add 15 mL or g Na biphenyl reagent, 
6.178(b), and swirl. If soln is not dark green, add more reagent. 
Let stand 3 min and add 3-5 mL H 2 dropwise. With stopper 
removed, swirl soln gently to decompose excess reagent. Add 
25 mL H 2 0, stopper, and mix with gentle rocking motion. (Do 
not shake vigorously.) Let layers sep. and drain lower aq. layer 
into 250 mL erlenmeyer. Re-ext solv. layer with two 25 mL 
portions 3/V HN0 3 and combine aq. solns in erlenmeyer. 

(b) Dusts, granular impregnates, and wettable powders. — 
Accurately weigh sample contg 0.5±0.05 g tech. chlordane into 
Soxhlet extn thimble. Ext with 80 mL benzene in Soxhlet app. 
1 hr. Transfer to 100 mL vol. flask, washing with several 3 mL 
portions benzene. Dil. to vol. with benzene and transfer 10 mL 
aliquot to 125 mL separator. Proceed as in (a). 



6.222 



Determination 



Add 15.00 mL 0.1/V AgN0 3 and 3 mL nitrobenzene to erlen- 
meyer, stopper, and shake vigorously 1 5 sec. Rinse stopper, add 
5 mL ferric indicator, 6.019(e), and back-titr. with 0.1/V KSCN to 
reddish-brown end point. (Designate mL KSCN as D.) 

Det. blank on reagents by pipetting 5 mL toluene into 125 mL 
separator, add 15 mL or g Na biphenyl reagent, and proceed as 
in 6.221(a), thru combining aq. solns in erlenmeyer. Add 15.00 



mL 0.1/V NaCI, 25.00 mL 0.1/V AgN0 3 , and 3 mL nitrobenzene, 
and proceed as above. Calc. blank correction factor, C = mL 
KSCN used in stdzn of AgN0 3 - mL KSCN used in blank detn. 

% Chlorine = [15 - (C + D) x F] x normality AgN0 3 x 3.545/ g 
sample. 

% Tech. chlordane = % CI x 1.56. 



Co/orimetric Method (62)— Official Final Action 

(Method is empirical; all conditions must be reproduced exactly 

to attain good precision. Temp., reaction time, and vol. of 

reagents affect color intensity.) 

6.223 Apparatus 

(a) Constant temperature bath. — Capable of maintaining 
100±1° and holding twelve 20 x 150 mm test tubes. 

(b) Cuvets. — 10 or 2 mm light path (available from Pyrocell 
Mfg. Co., 91 Carver Ave, PO Box 176, Westwood, NJ 07675). 

(c) Spectrophotometer.~Capab\e of accepting cuvets, (b). 



6.224 



Reagents 



(a) Methanol.— 90% (by vol.). 

(b) Methanol-benzene.—M\x 7 vols MeOH with 3 vols ben- 
zene. 

(c) Diethanolamine. — Purify by vac. distn at ca 20 mm Hg and 
take middle fraction. {Caution: See 51.011 and 51.015.) 

(d) Diethanolamine-KOH soln {Davidow reagent). — Mix 1 voL~ 
reagent (c) with 2 vols 1.0/V KOH in MeOH. 

(e) Chlordane std solns. — 1.5, 2.5, and 3.5 mg/mL Ref. grade 
(available from Velsicol Chemical Corp.). Dissolve tech. chlor- 
dane in reagent (b). Discard stds after 2 weeks. 



6.225 



Preparation of Sample 



(a) Liquid formulations. — Transfer weighed sample contg 
200-300 mg tech. chlordane to 100 mL graduate and dil. to 100 
mL with MeOH-benzene. 

(b) High concentration solid formulations {10% chlordane or 
more). — Treat as in (a) and shake vigorously several min. Let 
settle 1 hr. 

(c) Low concentration solid formulations {less than 10% chlor- 
dane). — Transfer weighed sample contg 200-300 mg tech. chlor- 
dane to Soxhlet and ext 1 hr with pentane. {Caution: See 51.039 
and 51.074.) Evap. pentane on steam bath and transfer ext to 
100 mL g-s graduate. Dil. to 100 mL with MeOH-benzene. 



6.226 



Determination 



Pipet 2 mL aliquot prepd sample to 20 x 150 mm test tube. 
Add No. 8 grit SiC boiling chip and 2 mL Davidow reagent, and 
place in 100° const temp. bath. Remove after exactly 45 min 
and cool immediately in beaker of cold H 2 0. Transfer to 10 mL 
vol. flask and dil. to vol. with 90% MeOH. Transfer aliquot of 
soln to 2 mm cell and read>4 at 550 nm within 15 min with 90% 
MeOH as ref. (Comparable results are obtained by dilg soln to 
50 mL and using 1 cm cell.) 

Treat 2 mL each std soln with each set of samples. (Read 1 std 
soln before samples, 1 after half the samples are read, and 1 
after last sample is read.) 

6.227 Calculations 

Calc. absorptivity {a) for each of 3 stds, and use av. in 
subsequent calcns (expected a is ca 0.25): a -A'/W, where 
A ' = A std soln and W - mg tech. chlordane (2 x concn std soln 
in mg/mL). 

% Tech. chlordane in sample = A x 5000/ (a x mg sample). 



98 



6. Pesticide Formulations 



AOAC Methods (1980) 



Hexachlorocyclopentadiene (HEX) 
(63)— Official Final Action 

(Applicable to tech. chlordane, but not to formulations} 

6.228 Reagent 

Hexachlorocyclopentadiene {HEX) std solns. — Stock soln. — 
0.1 g/100 mL Weigh 0.1000 g hexachlorocyclopentadiene Ref. 
Std (available from Velsicol Chemical Corp.) in 100 mL vol. flask, 
dil. to vol. with MeOH, and shake to dissolve. Std soln 1. — 0.005 
g/100 mL Dil. 5 mL stock soln to 100 mL with MeOH. Std soln 
2,— 0.002 g/100 mL Dil. 2 mL stock soln to 100 mLwith MeOH. 



Method I 



6.229 



Calibration 



With MeOH in both ref. and sample celts (matched 1 cm 
silica), adjust and 100% settings on UV spectrophtr at 324 nm. 
Empty sample cell, rinse several times with, and then fill with, 
std soln 1, and read A. Empty sample cell, rinse with MeOH, 
then rinse and fill with std soln 2, and read A Calc. A factor, K, 
for each std soln = (g std HEX/ 100 mL)/A Average the two K 
values. 



6.230 



Determination 



Weigh 0.5 g sample in 100 mL vol. flask, dil. to vol. with 
MeOH, and shake to dissolve. Proceed as in 6.229, treating 
sample soln in same manner as stds. 

% HEX in sample = {A of sample soln x 100 x /Q/(g 
sample/100 mL). 

6.231 Method II 

(Includes corrections for other components of 
chlordane which absorb at 324 nm) 

Proceed as in 6.229-6.230, except det. A of all solns at 300, 
324, and 350 nm. Settings of and 100% must be repeated at 
300, 324, and 350 nm for A readings at those points. Calc. K - 
(g std HEX/100 mL)/ [A 32A - 0.5(A 300 + A 350 )]. 

% HEX in sample = [4 324 - 0.b{A 300 + A 350 )] x 100 x K/{g 
sample/100 mL). 

AG Chlordane (Octachloro-4,7-methanotetra- 
hydroindane) (64) — Official Final Action 

a- and y-lsomers in Technical Products — Infrared Method 

(Not applicable to tech. chlordane or its formulations) 



6.232 



Apparatus and Reagent 

spectrophotometer. — Double beam, with 



(a) Infrared 
matched NaCI cells, 0.1 and 0.2 mm. 

(b) Std soln.— -Into tared 10 mL vol. flask, weigh 1.00±0.05 g 
ref. std a-chlordane and 0.38±0.02 g ref. std y-chlordane (Velsicol 
Chemical Corp.), dissolve in CS 2 , and dil. to vol. with CS 2 . 



6.233 



Preparation of Sample 



Melt entire sample in 100° oven and mix. Weigh 1.5±0.02 g 
into tared 10 mL vol. flask, dissolve in CS 2 , and dil. to vol. with 
CS 2 . 

6.234 Determination 

(a) a-Chiordane. — Fill ref. cell with CS 2 . Scan std and sample 
solns from 750 to 710 cm~ 1 (13.3 to 14.1 /Am), using 0.1 mm 
NaCI cells. Construct baseline from 738 to 715 cnrr 1 (13.6 to 14.0 
/Am) and draw line from point of max. A, ca 725 cm" 1 (13.8 /aiti), 
to intersect baseline. 



(b) y-Chlordane. — Fill ref. cell with CS 2 . Scan std and sample 
solns from 1390 to 1290 cnrr 1 (7.19 to 7.75 Atm), using 0.2 mm 
NaCI celis. Proceed as in (a), using min. at 1370 and 1310 cm -1 
(7.30 and 7.63 /Am), and det. A at max. 1320 cnrr 1 (7.58 /Am). 

6.235 Calculations 

Wt % a(a-chlordane) or y(y-chlordane) = {A x F x 100)/W S 
F - [W{a or y in std) x % purity in $\d]/A'{a or y of std), 

where W - wt (g), and A and A' refer to sample and std, resp. 
(Wts given are for cell thicknesses specified. For other cells, 

adjust wts to yield peak A between 0.2 and 0.5 (30-65% 7").) 

AG Chlordane in Granular Formulations — Infrared Method 

(Not applicable to tech. chlordane or its formulations. Caution: 
See 51.011, 51.041, 51.046, and 51.048.) 

6.236 Apparatus 

(a) Infrared spectrophotometer.— See 6.232. 

(b) Soxhlet extraction apparatus.— -With 25 x 80 mm What- 
man cellulose thimble. 

(c) Vigreux distilling tube. — 15 mm long. 

(d) Vials. — 5 dram, with plastic-lined screw caps. 

6.237 Reagents 

(a) Acetone. — Spectral grade. 

(b) Std soln.— Into tared 5 dram vial, weigh 1.00±0.05 g Ref. 
Std a-Chlordane and 0.38±0.02 g Ref. Std y-Chlordane (Velsicol 
Chemical Corp.) and pipet in 10 mL CS 2 . 



6.238 



Preparation of Sample 



Into tared thimble, weigh sample equiv. to wt of std and cover 
with glass wool. Insert into extn app. and attach to 250 mL flat- 
bottom $ flask contg boiling chips. Add 125 mL acetone, attach 
extractor to condenser, and ext 1 hr. 

Rinse extn app. with acetone. Sep. flask from extractor and 
condenser, attach distg tube to flask, and evap, acetone on 
steam bath. Remove tube, add 5 mL CS 2 , and evap. carefully. 
Repeat addn and evapn of CS 2 4 more times. (All residual 
acetone must be removed because acetone interferes with IR 
measurement.) Dry residue further, using forced air, until cryst. 
solid appears. Pipet 10 mL CS 2 into flask, and swirl carefully to 
dissolve solid. Release stopper pressure. 



6.239 

Proceed as in 6.234. 



Determination 



Calculation 



6.240 

Proceed as in 6.235 for calcn of wt %. 
% Total AG chlordane = % a-chlordane + % y-chlordane. 
Generally, factor representing specification grade of 95% AG 
chlordane may be used. Then, 

% AG chlordane = % total (« + y) x 1.053. 

Heptachlor in AG Chlordane — Gas 
Chromatographic Method 

(Not applicable to tech. chlordane or its formulations) 

6.241 Apparatus and Reagents 

(a) Gas chromatograph. — Equipped with flame ionization de- 
tector and 1.5 m (5') x %" id glass column packed with 5% 
silicone DC 200 (Viscosity 12500, Analabs, Inc.) on 130-140 mesh 



AOAC Methods (1980) 



DlMETHYLUREAS 



99 



Anakrom ABS. Operating conditions: temps (°) — column 165, 
injector 215, detector 220; N carrier gas 30 mL/min (ca 80 psig 
at inlet); and chart speed 0.5"/min. 

(b) Stds. — Ref. Std a-Chlordane, y-Chlordane, and Heptachlor 
(Velsicol Chemical Corp.) and hexachlorobenzene <C 6 CI 6 ) internal 
std (Eastman Kodak Co.), recrystd from benzene. 

(c) Std soln. — Accurately weigh following components into 
10 mL vol. flask, dissolve in CS 2 , and dil. to vol.: 0.48 g «- 
chlordane, 0.18 g y-chlordane, 0.010 g C 6 CI 6 , and 0.010 g 
heptachlor. 



6.242 



Preparation of Sample 



Melt entire sample in 100° oven and mix. Accurately weigh 
0.73 g sample and 0.010 g C 6 CI 6 into 10 mL vol. flask, and dil. 
to vol. with CS 2 . 

6.243 Determination 

Inject 1.5 fxL sample soln into gas chromatograph; retention 
times for C 6 CI 6 and heptachlor are ca 2 and 4 min, resp. Also 
inject 1.5 ptL std soln to det. response factor (/?f). 

Use attenuation (ca 2x) to keep internal std peak on scale and 
include in calcns. 

6.244 Calculations 

Calc. each peak area by any convenient means. Built-in 
integrators or planimeters provide most accurate method of 
detg areas where peaks are not perfectly symmetrical. 
% Heptachlor = {PH x RF x Wl x 100)/(/V x W) 
RF = {PI x W')/(PH' x Wl), 
where PH, PH', and PI = peak areas of sample, std heptachlor, 
and internal std, resp.; W, W, and Wl - g sample, std heptachlor, 
and internal std, resp. 

Chlorotoluron [3-{3-Chloro-4-methylphenyl)-1,1-dimethyl- 

urea], Chloroxuron [3-[4-(4-chlorophenoxy)phenyl]-1,1- 

dimethylurea], or Metoxuron (Dosanex- )[3-(3-Chloro-4- 

methoxyphenyl)-1,1-dimethylurea] (65) — Official Final Action 



CIPACAOAC Method 



6.245 



Principle 



Pesticide is extd from formulations with CH 2 CI 2 , free amines 
are removed with acid, and ext is hydrolyzed by alkali to Me 2 NH 
which is distd and titrd. Related byproducts, 3-(3-chloro-4-meth- 
ylphenylM-methylurea (I), 3-(4-methylphenyl)-1,1-dimethyiurea 
(II) (from chlorotoluron), 3-[4-(4-chlorophenoxy)phenyl]-1-meth- 
ylurea (III) and 3-(4-chlorophenyl)-1,1-dimethylurea (IV) (from 
chloroxuron), and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (V), 
3-(3-chloro-4-hydroxyphenyl)-1,1-dimethylurea (VI), and 3-(4- 
methoxyphenyl)-1,1-dimethylurea (VII) (from metoxuron), which 
may interfere, are detd by semiquant. TLC. Limit of detection 
for TLC is 0.1% for each byproduct. On same TLC plate for 
chloroxuron, free amine 4-(4-chlorophenoxy)aniline (VIM) is 
detd by sep. detection technic. Other byproducts, 1,3-bis(3- 
chloro-4-methylphenyl) urea (IX), 1,3-bis[4-(chlorophenoxy)- 
phenyl] urea (X), and 1 ,3-bis(3-chtoro-4-methoxyphenyl) urea (XI), 
do not interfere with chlorotoluron, chloroxuron, and metoxuron 
detns, resp. 

6.246 Preparation of Sample 

(a) Technical formulation. — Accurately weigh ca 3 g sample 
(4 g for chloroxuron) and transfer, using 100 mL CH 2 CI 2 , into 250 
mL separator, dissolve, and add 50 mL 1/V HCI. 

(b) Wettable powder. — Accurately weigh ca 3.5-4.0 g sample 



(for 80%) or 6.0-6.5 g (for 50%) into 200 mL beaker. Add 100 mL 
CH 2 CI 2 and stir mag. 5 min. Filter thru fritted glass crucible contg 
paper and 0.5 g layer of Celite, and rinse beaker and crucible 
with portions of CH 2 CI 2 to total vol. of ca 200 mL. Use only slight 
vac. to prevent crystn of pesticide on walls of crucible. Transfer 
quant, to 500 mL separator, and add 50 mL 1/V HCI. 



6.247 



Determination 



Vigorously shake mixt. 1 min and drain lower org. layer into 
second separator. Add 25 mL (50 mL for chloroxuron) 1/V HCI, 
shake 30 sec, and drain lower layer into 500 mL r-b flask. Wash 
the 2 acid layers successively with same 100 mL portion CH 2 CI 2 
(with two 50 mL portions for chloroxuron) and drain lower layer 
into the 500 r-b flask. Discard acid. 

Vac.-evap. CH 2 CI 2 in rotary evaporator to dryness at max. of 
40°. Remove all solv. to prevent interference in subsequent titrn. 
Add 100 mL propylene glycol, 40 g KOH, and some boiling 
stones to residue. Immediately connect flask securely to distn 
app. (Fig. 6:06) whose joints are lubricated with thin film of 
silicone grease. Place end of condenser delivery tube (sMO mm 
id) in 400 mL beaker below level of absorbing soln of 0.2 g 
H 3 B0 3 and 1 mL mixed indicator soln (40 mg methylene blue 
and 60 mg Me red dissolved in 100 mL alcohol) in 150 mL H 2 0. 
(To enhance end point, use 150 mL MeOH (2+1).) 

Gently warm flask until all particles dissolve; then boil 10 min 
or until propylene glycol distils into condenser. Titr. distd Me 2 NH 
continuously with stdzd 1/V HCI, 50.011-50.017. Complete distn 
by carefully adding H 2 dropwise from dropping funnel at rate 
of 1 drop/sec. Continue titrn until end point persists 2 min {V 
mL). Perform blank detn {B mL) with each series. 
% Pesticide = [(l/-B|xA/x F/g sample] 

- % byproducts (from 6.248), 
where F = 21.27 for chlorotoluron, 29.07 for chloroxuron, or 
22.87 for metoxuron, and N = normality of stdzd HCI. 




a heating bath or heating mantle 

b round bottom flask (500 ml) 

c distilling column, plain 

d dropping funnel {250 ml) 

e distilling head 

f distilling bridge 

g allihn condenser 

h beaker (400 ml) 

i magnetic stirrer 



/ooox 



FIG. 6:06 — Distillation apparatus (all dimensions in mm) 



100 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.248 Determination of Byproducts 

(a) For chlorotoluron. — Dissolve 100 mg each of byproducts 

1 and II (6.245) (available from Ciba-Geigy Ltd, Analytical De- 
partment, CH-4002 Basel, Switzerland) together in tetrahydro- 
furan and dil. to 50 mL in vol. flask. Dil. aliquots of 1, 2, 3, 4, and 
5 mL to 20 mL with tetrahydrofuran, equiv. to 0.2, 0.4, 0.6, 0.8, 
and 1.0%, resp., of each byproduct. 

Dissolve 1.0 g sample in tetrahydrofuran, and dil. to 20 mL 
with same solv. 

Spot 5 juL each of sample and std solns on 20 x 20 cm glass 
plates precoated with 0.25 mm layer of silica gel 60 F-254 (No. 
5715, E. Merck, Darmstadt, Germany, or equiv.), and develop by 
ascending technic in tank, presatd 30 min with developing solv. 
CHCI 3 -EtOAc (4+1), without filter paper linings, for ca 70 min 
(13 cm migration). Expose plate to 254 nm UV light and compare 
spots of samples with those of stds to est. concn of byproducts. 
Approx /? f values: chlorotoluron, 0.50; byproduct I, 0.25; by- 
product II, 0.35; and byproduct VI (does not interfere), 0.82. 

(b) For chloroxuron. — Dissolve 100 mg each of byproducts 
Ml, IV, and VIII (available from Ciba-Geigy Ltd) together in acetone 
and dil. to 100 mL in vol. flask. Dil. aliquots of 1, 3, 5, 8, and 10 
mL to 50 mL with acetone, equiv. to 0.1, 0.3, 0.5, 0.8, and 1% 
resp., of each byproduct. 

Dissolve 1.0 g sample in acetone, and dil. to 50 mL with same 
solv. Proceed as in (a), but use CHCI 3 -dioxane (9+2) as devel- 
oping solv. for ca 80 min (14 cm). Approx R f values: chloroxuron 
0.75; byproduct III, 0.40; byproduct IV, 0.65; and byproduct X 
(does not interfere), 0.90. 

Det. byproduct VIM on same TLC plate. Place beaker contg ca 

2 g NaN0 2 in empty developing tank and pour ca 3 ml HCI over 
salt. After 2 min, insert plate into tank 3 min, remove, and dry 
5 min at room temp, with hair dryer. Spray with 1% soln of N- 
(1-naphthyl)ethylenediamine.2HCI in 0.1/V HCI and compare 
violet sample spots with those of stds (/?,, 0.85). 

(c) For metoxuron. — Proceed as in (a), except use 100 mg 
each of byproducts V, VI, and VII (available from Sandoz Ltd, 
Agrochemical Division, Research, CH-4002 Basel, Switzerland). 
Approx. /? f values: metoxuron, 0.25; byproduct III, 0.34; by- 
product IV, 0.08; byproduct V, 0.13; and byproduct VII (does not 
interfere), 0.46. 



6.249 



Identification 



(a) Technical chloroxuron. — Record IR spectrum of 1 % CH 2 CI 2 
soln of sample and compare with spectrum of 1% CH 2 CI 2 soln 
of authentic ref. std. 

(b) 50% Wettable powder.— Stir ca 2 g sample and 2 g silica 
gel (70-230 mesh) with 100 mL CH 2 CI 2 5 min and percolate thru 
fluted filter. Record IR spectrum of filtrate in NaCI cell (0.5 mm 
path length) from 3000 to 650 cm -1 , using blank solv. as ref. 
Identity is established if sample spectrum corresponds qua), to 
that of std. 



Dichlobenil (2,6-Dichlorobenzonitrile) (66) 

CIPAC—AOAC Method 

6.250 Reagents and Apparatus 

(a) Dichlobenil.— -^99.5% purity (Philips-Duphar B.V., Weesp, 
Netherlands), or equiv. 

(b) Methyl myristate.— F\uka AG Cat. No. 70129, 5=99.5% 
purity {Fluka AG, Buchs, Switzerland), or equiv. 

(c) Mixed solvent soln. — 1,2-Dichloroethane-ether (1 + 1). 

(d) Internal std soln. — Dissolve 0.80 g Me myristate, (b), in 
100 mL mixed solv. soln, (c). 

(e) Calibration soln.— Accurately weigh ca 0.10 g dichlobenil, 



(a), into conical flask, pipet in 5 mL internal std soln, (d), and 
add 45 mL solv. soln, (c). 

(f) Gas chromatograph. — With on-column injection, flame 
ionization detector, injection port heating, and, preferably, de- 
tector heating. Pyrex column 1.83 m x 3 mm id, packed with 
10% Carbowax 20M on 100-120 mesh Chromosorb P, acid 
washed, dimethyldichlorosilane treated (available from Analabs, 
Cat. No. GCP-G09D). Operating temps (°): oven 200, injection 
port 210, detector 210. Carrier gas (N) flow rate 25 mL/min. 
Approx. retention times 7 and 12 min for Me myristate and 
dichlobenil, resp. 

6.251 Preparation of Sample 

(a) Technical dichlobenil. — Accurately weighca2.0gdichlob- 
enil into 100 mL vol. flask. Dissolve in mixed solv. soln, (c), and 
dil. to vol. Pipet 5 mL aliquot into 100 mL conical flask, add 5.00 
mL internal std soln, (d), and dil. to 50 mL with mixed solv., (c). 

(b) Wettable powders. — Accurately weigh sample contg ca 
1 .0 g dichlobenil into 100 mL vol. flask, add few mL mixed solv., 
(c), swirl, and dil. to vol. with mixed solv. Let settle, pipet 10 mL 
clear supernate into 100 mL conical flask, and continue as in (a). 

(c) Granules. — Accurately weigh ca 6.0 g sample into 100 mL 
conical flask. Add 20 mL dichloroethane and stir 10 min on mag. 
stirrer. Filter with vac. thru glass filter paper (Whatman GF 82, 
or equiv.), supported on fritted glass filter. Wash granules 5 
times with 5 mL dichloroethane, collect filtrate in 100 mL vol. 
flask, and dil. to vol. with ether. Pipet aliquot of this soln, contg 
ca 0.1 g dichlobenil (10 mL for 20% granules, 25 mL for 7% 
granules), into 100 mL conical flask, add 5.00 mL internal std 
soln, (d), and dil. to 50 mL with mixed solv., (c). 



6.252 



Determination 



Inject 2 /u,L portions of calibration soln, (e), until response 
factor varies <1% for successive injections. Inject duplicate 2 
Hi portions of sample soln, followed by 2 /aL portions of 
calibration soln, (e). Measure peak areas of dichlobenil and Me 
myristate, either by multiplying peak ht by retention time, or by 
digital integration. Use av. of duplicate values. 



6.253 



Calculation 



p = u q x r x 20)/{/ r x q), 
where p = response factor, l q and l r - peak areas of internal std 
and dichlobenil, resp., q = g internal std, r = g dichlobenil in 
calibration soln. (Response factor is ca 1.5.) 

% Dichlobenil = {f d xqxpxFx 100)/(l m x W x 20), 
where l d and l m = peak areas of dichlobenil and internal std, 
resp., p = response factor, F - diln factor for sample (100/x, 
where x = mL taken to obtain final soln), and W = g sample. 



Dichlorodiphenyltrichloroethane (1,1,1-Trichloro-2,2- 
bis(p-chlorophenyl) Ethane) (DDT) — Official Final Action 

it Totai Benzene-Soluble Chlorine Method [67) * 

(Applicable in absence of other org. CI compds. Use H 2 2 and 
isoamyl alcohol-ether extn method on dispersible powders or 
sprays that contain surface active agents or other ingredients 
that react with AgN0 3 . Caution: See 51.034, 51.039, 51.040, 
51.041, and 51.045.) 

6.254 Reagents 

(a) Benzene.— Thiophene- and Cl-free. 

(b) Metallic sodium. — Ribbons or small pieces. 



ir Surplus method— see inside front cover. 



AOAC Methods (1980) 



DDT 



101 



(c) Decolorizing carbon. — Test for presence of CI by heating 
with HN0 3 (1+4), filtering, and adding AgN0 3 soln to filtrate. If 
CI is present, wash with the HN0 3 until washings are Cl-free. 

Note: Det. blank on all reagents, limiting 0.1/V AgN0 3 to 5 mL. 



6.255 



Preparation of Solution 



(a) Technical grade DDT. — Weigh sample contg ca 1 g DDT 
and transfer to 250 ml_ vol. flask. Dissolve sample in 10 mL 
benzene; then dil. to vol. with 99% isopropanol. Transfer 25 
mL aliquot to 250-500 mL f erlenmeyer. (Direct weighing of 
sample may be substituted, provided it does not introduce error 
>0.1%.) 

Add 2.5 g Na and shake to mix sample with isopropanol. Do 
not add Na thru top of condenser or get Na on ground glass 
joints. Connect flask to reflux condenser and boil gently ^30 
min, shaking occasionally. Eliminate excess Na by cautiously 
adding 10 mL 50% isopropanol thru condenser at rate of 1-2 
drops/sec. Disconnect condenser, add 60 mL H 2 0, boil soln ca 
30 min to expel isopropanol, and proceed as in 6.256(a), (b), (c), 
or (d). 

(b) Dusting mixtures containing DDT in absence of organic 
matter, — Weigh sample contg ca 0.75 g DDT, transfer to 100-200 
mL vol. flask, and add exactly 100 mL benzene. Shake until DDT 
dissolves and soln is well mixed. Let settle and transfer 10 mL 
aliquot to 250-500 mL f erlenmeyer. 

Evap. on steam bath to remove most of benzene. (Do not 
evap. to dryness, as DDT may decompose with loss of HCI.) 
Add 25 mL 99% isopropanol and proceed as in (a), second par. 

If free S is present, proceed as in (f), beginning 'Then add 5 
mL30%H 2 O 2 " 

(c) Dusting mixtures in presence of organic matter {coloring 
matter, plant resins, etc.). — Weigh sample contg ca 0.75 g DDT, 
transfer to 100-200 mL vol. flask, and add 0.5-1.0 g decolorizing 
C and exactly 100 mL benzene. Shake until DDT dissolves and 
soln is well mixed. Filter into narrow-neck flask thru fast qual. 
paper without suction, keeping funnel covered with watch glass 
to avoid evapn loss. Transfer 10 mL aliquot to 250-500 mL f 
erlenmeyer. Proceed as in (b), second par. Before detg CI remove 
org. matter as follows: 

Cool, add 2-3 drops phthln, and neutze by adding HN0 3 (1+1) 
dropwise; then 10 mL excess. Cool, if necessary, to room temp., 
transfer contents of flask and aq. washings to small separator, 
and shake with 15 mL isoamyl alcohol-ether (1 + 1). Drain aq. 
layer into second separator and ext again with 15 mL isoamyl 
alcohol-ether (1 + 1). Drain aq. layer into 250 mL beaker. Wash 
the 2 exts successively with two 10 mL portions H 2 0. Combine 
aq. wash solns with aq. soln in beaker. Det. CI by one of 
following methods: 

{1) Proceed as in 6.256(a), beginning "Add slight excess 0.1/V 
AgN0 3 , . . ." 

{2) Proceed as in 6.256(b), beginning "Add 0.1/V AgN0 3 . . ." 

(3) Proceed as in 6.256(c), beginning "Cool flask to room 
temp. ..." 

(4) Add 2-3 drops phthln to sample, make alk. by adding 1/V 
NaOH, and proceed as in 6.256(d), beginning ". . . transfer 
contents to Pt dish." 

(d) Mineral oil sprays in absence of organic matter {plant 
extractive material, organic thiocyanates). — Transfer weighed 
sample contg 0.06&-O.075 g DDT to 250-500 mL f flask. Add 25 
mL 99% isopropanol and proceed as in (a), second par. 

Note: If DDT content is <2%, use isoamyl alcohol-ether extn, 
(c), second par., to remove excess oil. 

Proceed as in 6.256(a), (b), (c), or (d). 

(e) Mineral oil sprays in presence of organic matter {plant 
extractive material from pyrethrum or derris and /or cube.) — 



Proceed as in (d), using isoamyl alcohol-ether extn, (c), to 
remove excess oil. 

(f ) Mineral oil sprays in presence of organic thiocyanates with 
or without plant extractive material. — Transfer sample contg 
0.065-0.075 g DDT to 250-500 mL f erlenmeyer. Add 25 mL 
99% isopropanol and proceed as in (a), second par., thru ". . . 
add 60 mL H 2 0, . . ." Then add 5 mL 30% H 2 2 , few drops at 
time, thru top of condenser, heat mixt. in flask to bp, and boil 
15 min. Add addnl 5 mL H 2 2 and again boil 15 min. Add 15 mL 
more H 2 2 , disconnect reflux condenser, and boil 15-30 min to 
expel isopropanol. Proceed as in (c), second par. 

(g) Emulsions {solvent, emulsifier, and water). — Weigh well 
mixed sample contg ca 0.75 g DDT in weighing bottle. Wash 
into 100 mL vol. flask and dil. to vol. with isopropanol. Transfer 
10 mL aliquot to 250-500 mL $ erlenmeyer. Expel isopropanol 
and H 2 on steam bath in air current. If drops of H 2 still remain, 
add 10 mL isopropanol and repeat evapn. Add 25 mL 99% 
isopropanol and proceed as in (a), second par. 

Note: If S is brought into the soln as by decomposition of 
emulsifier, proceed as in (f), beginning "Then add 5 mL 30% 
H 2 2l . . r 



6.256 



Determination 



(a) Cool flask and transfer contents to 250 mL beaker. Add 
2-3 drops phthln and neutze with HN0 3 (1+1); then add 10 mL 
excess. Add slight excess 0.1/V AgN0 3 , 50.031, and coagulate 
pptd AgCI by digesting on steam bath 30 min, stirring frequently. 
Cool, filter thru fast qual. paper, and wash thoroly with H 2 0. 
Add 5 mL satd ferric indicator, 6.019(e), and det. excess AgN0 3 
in filtrate by titrn with 0.1/V KSCN, 50.030(b). Subtract amt AgN0 3 
found in filtrate from that originally added. Difference is that 
required to combine with CI in the DDT. 1 mL 0.1/V 
AgN0 3 - 0.003545 g CI. CI x 2 = DDT. 

(b) Cool flask, add 2-3 drops phthln soln, and neutze with 
HNO3 (1 + 1); then add 10 mL excess. Add 0.1/V AgN0 3 from 
buret in excess of amt necessary to ppt all CI; then add 5 mL 
nitrobenzene and 0.5 g fe 2 (S0 4 ) 3 and swirl flask to coagulate 
ppt. Back-titr. excess AgN0 3 with 0.1/V KSCN to faint pink. Cross- 
titr. with both std solns, crossing end point in each direction to 
assure results. From vol. AgN0 3 , calc. % DDT as in (a). 

(c) Cool flask, add 2-3 drops phthln, neutze with HN0 3 (1+1), 
and add 6 mL excess. Cool flask to room temp, and transfer 
contents to 400 mL beaker. (Vol. should be 200-250 mL) Titr. 
CI with 0.1/V AgN0 3 potentiometrically, using Ag-AgCI electrodes 
(Fisher Titrimeter, or equiv.). Calc. % DDT as in (a). 

Note: When this method is used, decolorizing C step in 
6.255(c), and isoamyl alcohol-ether extn in 6.255(c), (d), and (e), 
may be omitted. 

(d) Cool flask and transfer contents to Ptdish. Evap. to dryness 
and ignite as thoroly as possible at =£525°. Ext with hot H 2 0, 
filter, and wash. Return residue to Pt dish and ignite to ash; 
dissolve in HN0 3 (1+4), filter from any insol. residue, wash 
thoroly, and add this soln to aq. ext. Add 0.1/V AgN0 3 , avoiding 
more than slight excess. Heat to bp, protect from light, and let 
stand until ppt coagulates. Filter on weighed gooch, previously 
heated to 140-150°, and wash with hot H 2 0, testing filtrate to 
prove excess of AgN0 3 . Dry AgCI at 140-150°, cool, and weigh. 
Calc. % CI and DDT as in (a). 

infrared Method {68) 

[Caution: See 51.041.) 
6.257 Reagent 

DDT std soln.— Weigh 0.250 g tech. DDT into 50 mL vol. 
flask or g-s container and add exactly 25 mL CS 2 . If sample to 
be analyzed contains S, add wt of S expected in portion of 



102 



6. Pesticide Formulations 



AOAC Methods (1980) 



sample to be taken for analysis. Shake to dissolve and add small 

amt anhyd. Na 2 S0 4 . Centrf. portion of soln if it is not clear. 

6.258 Determination 

Weigh sample contg ca 0.25 g DDT into 50 mL vol. flask and 
add exactly 25 mL CS 2 and small amt anhyd. Na 2 S0 4 . Let stand 
5=30 min with occasional shaking. Transfer portion to g-s test 
tube and centrf. short time. Transfer to NaCI cell and scan with 
infrared spectrophtr, using 0.5 mm cell in region, 8.5-10.5 yxm. 

Scan std soln in same manner. 

Measured of DDT peak at 9.83 /nm with baseline from 9.4 to 
10.2 ^m, and calc. % DDT, 



Dimethyl 2,3,5,6-Tetracnloroterephthalate 
(Dacthal) (69)— Official Final Action 

(Caution: See 51.011, 51.018, 51.039, 51.040, 
51.041, 51.045, 51.046, and 51.048.) 

Gas Chromatographic Method 

{Under conditions specified, other pesticides or ingredients may 
interfere with GLC analysis, e.g., aldrin has same retention time 
as Dacthal. Aldrin and Dacthal may be sepd at 170° column 

temp.) 



6.259 



Apparatus 



Gaschromatograph. — 1.8 m (6') x %" id stainless steel column 
contg 10% silicone UC-98 (Applied Science Laboratories, Inc.) 
on 80-100 mesh silanized Diatoport S (Hewlett-Packard Co., Rt 
41, Avondale, PA 19311). Conditions (applicable to Hewlett- 
Packard F&M Model 5750) — temps(°): column 200, injection port 
240, flame ionization detector 260; H, air, and He carrier flows, 
115, 600, and 25 mL/min, resp.; chart speed 0.25"/min; atten- 
uation 4x; range setting 10 2 (10~ 10 amp full scale). 



6.260 



Preparation of Standard Curve 



(a) Dacthal std solns.— Weigh 0.5 g Dacthal (available from 
Diamond Shamrock Corp., PO Box 348, Painesville, OH 44077) 
into 100 mL vol. flask, add ca 90 mL acetone (soln is rapid), and 
dil. to vol. Pipet 5, 10, and 15 mL into sep. 25 mL vol. flasks and 
dil. to vol. with acetone. 

(b) Hexachiorobenzene [HCB) std solns.— -Weigh 0.5 g ref. 
grade HCB into 100 mL vol. flask, add 90 mL benzene, and dil. 
to vol. with benzene. Pipet 1, 2, and 3 mL into sep. 25 mL vol. 
flasks and evap. to dryness with current of dry air. Add 20 mL 
acetone to each flask and dil. to vol. with acetone. 

Inject 5 |U.L each dild HCB and Dacthal std at least twice. Prep, 
curve of peak area or ht against concn for Dacthal and peak ht 
against concn for HCB. 



6.261 



Determination 



(a) Benzene extraction— Grind granular product. Weigh por- 
tion contg ca 300-400 mg Dacthal into Whatman extn thimble 
(33 x 88 mm). Cover with glass wool. Place thimble in medium 
Soxhlet extractor; add 150-175 mL benzene and 3 glass beads. 
Ext 6 hr. Quant, transfer ext to 400 mL beaker and evap. to ca 
5 mL on steam bath with dry air current; remove and evap. to 
dryness with air current. Add ca 150 mL acetone and let stand 
until soln is complete (white, flaky crystals may indicate incom- 
plete soln; soln may be hastened by placing flask in ultrasonic 
cleaner). Filter soln thru glass wool into 200 mL vol. flask. Wash 
beaker with acetone, transfer washings to vol. flask, and dil. to 
vol. 

(b) Alternative acetone extraction. — Substitute acetone for 



benzene in extn. Proceed as in (a) thru "Ext 6 hr." Continue with 
"Filter soln thru glass wool . . ." 

Inject duplicate 5 tiL sample soln into gas chromatograph. 
Compare peak ht or peak area to std curve to det. % hexa- 
chiorobenzene (HCB) and Dacthal. 



Infrared Method 



6.262 



Preparation of Sample 

Grind granular product. Weigh sample contg 200-500 mg 
Dacthal into Whatman extn thimble. Proceed as in 6.261(a) thru 
". .'. evap. to dryness with air current." Add 25 mL CS 2 , allow 
ca 30 min for complete soln, and transfer quant, to 50 mL vol. 
flask with CS 2 , filtering sample thru glass wool. Dil. to vol. 



6.263 



Preparation of Standard Solution 



Weigh 1.25 g Dacthal into 100 mL vol. flask. Add ca 90 mL CS 2 
(soln may be hastened by placing flask in ultrasonic cleaner) 
and dil. to vol. Pipet 10, 15, and 20 mL into sep. 25mLvol. flasks 
and dil. to vol. 



6.264 



Determination 



Set spectrophtr at optimum operating condition. Use 0.5 mm 
KBr (or NaCI) matched cells. Fill ref. cell with CS 2 . Transfer dild 
stds to other cell and scan slowly from 1 100 to 900 cm -1 . Repeat 
with samples. Construct baseline from 1030 to 925 crrr 1 and 
draw line from midpoint of max. A at ca 964 cm -1 to intersect 
baseline. Compute A/4 at 964 cm -1 at point of intersection of 
stds and sample. 

Prep. AA -concn curve for std; Beer's law is obeyed over concn 
range 2-15 mg Dacthal/ mL. Calc. % Dacthal from std curve. 



Dicamba (3,6-Dichloro-o-anisic Acid; 2-Methoxy-3,6- 
dichlorobenzoic Acid) (70)— Official Final Action 

6.265 Reagents and Apparatus 

(a) Acetone. — Spectral grade. 

(b) Dimethylamine [DMA) soln.— -60% (w/w). 

(c) Dicamba std. — Ref. grade (Velsicol Chemical Corp.). 

(d) Infrared spectrophotometer. — With BaF 2 cells, 0.025 mm, 
and matched NaCI cells, 0.2 mm. 

6.266 Preparation of Sample 

(Sample wts are for cell thicknesses specified. For other cells, 
adjust wts to yield peak between 30 and 60% 7".) 

(a} Aqueous solns of DMA salt (4 lb /gal. ). — Pipet, using same 
pipet as for std, 5.00 mL sample into tared 25 mL vol. flask and 
weigh, Dil. to vol. with acetone. (Use this soln directly in 0.025 
mm BaF 2 cell.) 

(b) Solns of DMA salt {other concentrations). — Prep, as in 
(a), adjusting sample size to yield 2.4 g dicamba/25 mL. 

(c) Technical dicamba. — Weigh 0.2±0.005 g sample into tared 
25 mL vol. flask and dil. to vol. with CS 2 . 



6.267 



Preparation of Standard 



(a) Liquid formulations. — (7) Aqueous solns of DMA salt {4 
lb / gal.): Weigh 11.98±0.02 g dicamba std into tared 50 mL 
beaker. Add 5 mL H 2 and 4 mL 60% DMA. Adjust pH to 7.0 by 
titrg with 60% DMA soln, using mag. stirrer and pH meter. (All 
solids should be dissolved at this time.) Rinse each pH electrode 
with two 1 mL H 2 rinses (4 mL total), collecting rinses in the 
50 mL beaker. Cool soln to room temp, and transfer to tared 25 
mL vol. flask. Rinse beaker twice with H 2 0, collecting rinses in 



AOAC Methods (1980) 



2,4-D 



103 



flask. Dil. to vol. with H 2 and mix thoroly. Weigh flask and 
contents to det. total wt of soln. Pipet 5.0 ml_ std formulation 
into tared 25 mL vol. flask, weigh, and dil. to vol. with acetone. 

{2) Aqueous sofns of DMA salt {other concentrations): Prep, 
as in (a)(7), adjusting dicamba content to required concn. 

(b) Technical dicamba. — Weigh 0.2±0.005 g dicamba std into 
tared 25 mL vol. flask and dil. to vol. with CS 2 . 



6.268 



Determination 



(a) Liquid formulations. — Record spectra of std and sample 
between 1070 and 930 cm" 1 (9.3-10.7 /xm), using BaF 2 cell. Use 
air in ref. beam. Obtain AA and AA' for sample and std, resp., 
at 1012 cm -1 (9.89 /xm) from horizontal baseline tangent to min. 
between 1020 and 1070 cm -1 (9.4-9.7 /xm). 

(b) Technical dicamba. — Record spectra of std and sample 
from 1100 to 930 cm" 1 {9.1-10.7 /xm), using NaCI cells. Use CS 2 
in ref. cell. Obtain AA and AA ' for sample and std, resp., at 1012 
cnrr 1 (9.89 /xm) from horizontal baseline tangent to min. between 
1075 and 1035 cm" 1 (9.3-9.66 /Am). 



6.269 



Calculations 



(a) Liquid formulations. — Dicamba, lb/gal. = AA x CjAA', 
where C = lb std/gal. = (g std x % purity of std x 8.35)/25. 

% Dicamba by wt = {AA x F)/(g sample/25 mL), where F = 
[(g std/25 mL) x % purity of std]/ AA'. 

(b) Technical dicamba. — % Dicamba by wt = A A x F/g 
sample, where F = (g std x % purity of std)/AA'. 



Dicamba~2-Methyl-4-chlorophenoxyacetic Acid (MCPA) 
and Dicamba-2,4-D (7/)— Official Final Action 



AOACdPAC Method 

{Caution: See 51.041.) 



6.270 



Principle 

Method is applicable to aq. dimethylamine (DMA) salt for- 
mulations of dicamba and 2-methyl- 4-chlorophenoxyacetic acid 
(MCPA) or 2,4-D (2,4-dichlorophenoxyacetic acid). Active ingre- 
dients are pptd by HCI and extd with CHCI 3 . Solv. is evapd, 
residue dissolved in acetone, and>4 measured at characteristic 
IR wavelengths. 

6.271 Preparation of Standard Solutions 

(a) Dicamba-MCPA. — Accurately weigh 0.20 ±0.02 g dicamba 
and 0.60±0.02 g MCPA into tared weighing bottle. Pipet in 25 
mL acetone and swirl until completely dissolved. If cells other 
than 0.2 mm are used, adjust wts to g\veA of 0.2-0.5 (30-65% 
T) for both std and sample solns. 

(b) Dicamba-2,4-D—Prep. as in (a), using 0.20±0.02 g di- 
camba and 0.40±0.02 g 2,4-D. 

6.272 Preparation of Sample 

(a) Dicamba-MCPA. — Accurately weigh sample contg 
0.20±0.02 g dicamba and 0.60±0.02 g MCPA into tared weighing 
bottle. Add 5 mL H 2 and transfer quant, to 125 mL separator 
with 5-10 mL H 2 0. 

(b) Dicamba-2,4-D.— Prep, as in (a), using 0.20±0.02 g di- 
camba and 0.40+0.02 g 2,4-D. 



6.273 



Determination 



ppt. Drain CHCI 3 ext into 1 25 mL erlenmeyer and re-ext with two 
15 mL portions CHCI 3 . Add boiling chips to combined ext and 
evap. on steam bath to dryness. Let dry in hood overnight at 
room temp. (Do not dry in air or vac. oven.) Pipet in 25 mL 
acetone and swirl to completely dissolve residue. Add few g 
granular anhyd. Na 2 S0 4 if any H 2 is present, 

Record IR spectrum and measure AA in matched 0.2 mm NaCI 
cells with acetone in ref. cell at following wavelengths: 

(a) Dicamba-MCPA.— Range, 1135-930 crrr 1 (8.8-10.75 /xm); 
dicamba peak, 1012 cm" 1 (9.89 /xm); MCPA peak, 1070 cm" 1 
(9.35 /xm); baseline, horizontal tangent to min. at 970-965 cnrr 1 
(10.3-10.4 /xm) for both constituents. 

(b) Dicamba-2,4-D.— -Range, 1130-945 cnrr 1 (8.85-10.6 /xm); 
dicamba peak, 1012 cnrr 1 (9.89 /xm); 2,4-D peak, 1080 cm" 1 (9.26 
/xm); baseline, horizontal tangent to min. at 970-960 cm" 1 
(10.3-10.4 /xm) for both constituents. 



6.274 



Calculations 



To soln add HCI dropwise with const swirling to pH 1; then 
add 5 drops excess. Pipet in 25 mL CHCI 3 and shake to dissolve 



% by wt of constituent - {AA/W){W x P/AA'), where AA 
and AA' = absorbance of constituent in sample and std solns, 
resp.; W and W — g constituent in sample and std solns, resp.; 
and P — % purity of constituent in ref. std. 

lb/gal. = % by wt x sp gr x 8.345. 

2,4-D (2,4-Dichlorophenoxyacetic Acid) 

Automated High Pressure Liquid Chromatographic 
Method [72)— Official First Action 

6.275 Principle 

Esters of 2,4-D are saponified in situ; amine salts are converted 
to H 2 0-sol. K salt of 2,4-D. Ionic 2,4-D is protonated by pH 2.95 
CH 3 CN-H 2 (1+4) eluant, and sepd from all known impurities 
and p-bromophenol internal std on reversed phase bonded 
microparticulate column. 2,4-D elutes between impurities 2,4- 
and 2,6-dichlorophenol. 

6.276 Apparatus 

{a) Liquid chromatograph. — Fitted with 5000 psi pressure 
gage, 280 nm UV detector, line filter in eluant reservoir, and 10 
mv full scale deflection strip chart recorder. Automated sampling 
system and computing integrator are optional. Typical operating 
conditions: chart speed, 0.2 cm/min; eluant flow rate, 0.9-3.0 
mL/min depending upon psi range of pump; detector sensitivity, 
0.64/4 unit full scale; temp., ambient; injection valve vol., 10 /xL. 

(b) Liquid chromatographic column. — No. 316 stainless steel, 
250 x 4.6 (id) mm, Partisil® 10 /xm ODS column with 50 x 4.6 
(id) mm Co:Pell ODS pellicular guard column (Nos. 6526-124 
and 6561-404, resp., Whatman Inc., 9 Bridewell PI, Clifton, NJ 
07014). Regenerate, if necessary, by pumping CH 3 CN thru col- 
umn until baseline is stable. Repack first 5 mm of guard and 
main columns with Co:Pell ODS if peaks begin to "tail". 

6.277 Reagents 

(a) Eluant— pH 2.95. CH 3 CN (distd-in-glass)-H 2 (deionized, 
0.4 /tm filtered) (1+4) contg NaOH added from (1+1) aq. soln 
of known normality, 50.033(b), at final vol. concn of 0.3/W. Add 
H3PO4 to adjust pH to 2.95. 

(b) Saponification-internal std soln. — 4 g p-Bromophenol/L 
0.2/V KOH in isopropanol-H 2 (2 + 1). Add KOH from (1 + 1) aq. 
soln of known normality. 

(c) 2,4-D std soln.— 300 mg/25 mL. Accurately weigh ca 300 
mg 2,4-D anal. ref. std (99+% isomer pure; available from Dow 
Chemical Co., Sample Coordinator, 9001 Bldg, Midland, Ml 
48640), previously dried 15 min at 100°, into 1 or 2 oz glass vial 



104 



6. Pesticide Formulations 



AOAC Methods (1980) 



with polyethylene-lined screw cap. Pipet in 25 ml_ saponification- 
internal std soln, and shake to dissolve. Prep. 2,4-D std soln and 
sample soln, 6.278, at same time, using same pipet. 

6.278 Preparation of Sample 

Accurately weigh sample contg ca 300 mg 2,4-D acid equiv. 
into 1 or 2 oz glass vial with polyethylene-lined screw cap. Pipet 
in 25 ml_ saponification-internal std soln, and shake 15 min, 
warming ester formulations to 50° several min before shaking. 
Filter prepd sample thru 9 cm Whatman glass microfiber filter 
GF/A, or equiv., collecting major portion of aq. phase for 
chromatography. Adjust isopropanol-H 2 ratio, if necessary, to 
obtain complete dissoln of sample; e.g., amine formulations are 
best prepd with isopropanol-H 2 (1+1). 



6.279 



Determination 



Transfer ca 1 mL portions of samples and stds to automated 
sampler vials, and cap. Place samples and stds in position, and 
start automatic sampler. With programmed integrator use fol- 
lowing calcn program automatically: 

% 2,4-D - (/?//?') x (W'/W) x P, 

where R and R' = peak ht or area ratios of 2,4-D to internal std 
for sample and std, resp.; W = mg 2,4-D in std; W = mg 
sample; and P = % purity of std. If automated sampler and 
computing integrator are unavailable, inject 10 pX. samples and 
stds and perform calcns manually. As check on calibration, place 
stds in sample sequence at beginning, middle, and end. Period- 
ically confirm linearity by analyzing stds contg 200, 300, and 
400 mg 99 + % 2,4-D/25 mL saponification-internal std soln. 
Continuously recycle and mag. stir eluant. Replace eluant after 
ca 200 injections/L 



Sodium Salt of Dalapon (2,2-Dichloropropionic 
Acid) {73)— Official Final Action 

{Caution; See 51.041.) 

6.280 Apparatus 

(a) Reflux apparatus. — 250 mL erlenmeyer connected thru £ 
35/25 ball joint to reflux condenser. 

(b) Filtering apparatus. — 60 mL, medium porosity fritted glass 
funnel attached to glass filter bell, 11 cm od, 18 cm high, with 
bottom gasket and slide valve. 

6.281 Reagents 

(a) Mercuric-cupric nitrate soln. — [Caution: See 51.065.) Dis- 
solve 100.0 g yellow HgO and 60 g Cu(N0 3 ) 2 .3H 2 in 500 mL 
3.100±0.003/V HN0 3 , measured from vol. flask, in 1 L vol. flask, 
dil. to vol. with H 2 0, and filter. 

(b) Potassium iodide soln. — Dissolve 150 g Kl in H 2 0, dil. to 
1 L, and neutze to phthln. 



6.282 



Determination 



Accurately weigh sample contg 0.11-0.22 g Na salt of 2,2- 
dichloropropionic acid, transfer to erlenmeyer of reflux app., 
and add 100 mL Hg-Cu nitrate soln. Add some boiling chips, 
attach condenser, and reflux 15 min. Cool in H 2 bath. Filter 
thru filtering app., washing flask and ppt acid-free with H 2 from 
wash bottle. Discard filtrate and washings, and place 250 mL 
narrow-mouth erlenmeyer in filtering bell. 

Add 50 mL Kl soln to erlenmeyer to dissolve any remaining 
ppt, transfer to funnel, and stir until ppt dissolves. Draw soln 
into narrow-mouth erlenmeyer with vac. Wash flask and funnel 
with ^50 mL Kl soln from wash bottle, adding washings to 



filtrate. Add few boiling chips to filtrate and boil 1 min. Cool in 
H 2 bath. Titr. immediately with 0.1/V HCI, using phthln. 

% Na salt 2,2-dichloropropionic acid = mL 0.1/V HCI x 0.004499 
x 100/g sample. 



Dicofol (Kelthane®, 4,4 -Dichloro-«-(trichloromethyl)- 
benzhydrol) 

Potentiometric Method [74) — Official First Action 

6.283 Principle 

Dicofol is hydrolyzed in ale. KOH under reflux, and hydrolyz- 
able org. CI is converted to ionizable CI which is titrd potentio- 
metrically with std AgN0 3 . 



6.284 



Apparatus 



(a) Condenser. — f 24/40 Pyrex condenser, water cooled, 400 
mm long with drip tip. 

(b) Potentiometer.— Fisher Ace u met Model 320 (new model 
325) expanded scale pH meter, or equiv., with 50 mL buret 
graduated in 0.1 mL r Ag billet indicating electrode (Fisher No. 
13-639-122), and Ag-AgCI ref. electrode (Fisher No. 13-639-53). 
Keep Ag electrode free from tarnish by polishing with aq. 
NaHC0 3 -CaC0 3 (1 + 1) paste. Before each analysis, rinse Ag 
electrode with NH 4 OH (1 + 1) followed by H 2 0. 



6.285 



Reagents 



(Use deionized H 2 thruout.) 

(a) Alcoholic potassium hydroxide soln. — 0.5/V. Dissolve 28.1 
g KOH pellets in ca 600 mL alcohol and dil. to 1 L with alcohol. 

(b) Potassium chloride std soln. — 0.1/V. Dissolve 7.456 g KCI 
in H 2 and dil. to 1 L with H 2 0. 

(c) Silver nitrate std soln.— 0.1/V. Dissolve 17.00 g AgN0 3 in 
100mLH 2 O, add 1.7 mL HN0 3 , and dil. to 1 L with H 2 0. To stdze, 
dil. 25 mL 0.1/V KCI to 200 mL with H 2 in 400 mL beaker. Adjust 
pH to 2.0+0.2, using NH 4 OH (1+4) and/or HN0 3 (1+4), and dil. 
to 300 mL. Titr., using potentiometer as in 6.288. Plot mv against 
vol. 0.1/V KCI and det. mL 0.1/V AgN0 3 at end point. Calc. 
normality of AgN0 3 std soln. Stdze AgN0 3 std soln daily. (Equiv. 
wt dicofol - 370.5/3 = 123.5.) 

(d) Thymol blue indicator soln. — 0.1%. Dissolve 100 mg thy- 
mol blue in 100 mL alcohol (1 + 1). 



6.286 



Preparation of Sample 



(a) Kelthane technical. — Fuse sample in loosely capped jar in 
100 c oven and mix thoroly with glass rod. Accurately weigh ca 
4-6 g molten sample into 150 mL beaker. Add 50-75 mL 
isopropanol and heat with occasional swirling until sample 
dissolves. Transfer quant, to 500 mL vol. flask, let cool to 25°, 
and dil. to vol. with isopropanol. Pipet 25 mL sample soln into 
300 mL f 24/40 Pyrex erlenmeyer. {Caution: See 51.018.) 

(b) Kelthane formulations. — (7) Kelthane MF and Kelthane 
35. — Accurately weigh ca 1 g sample into 300 mLf 24/40 Pyrex 
erlenmeyer. (2) Kelthane EC. — Proceed as in (7), using ca 2 g 
sample. 

6.287 Hydrolysis 

Transfer 50 mL ale. KOH soln to erlenmeyer contg sample. 
Attach condenser, seal with 2-3 drops alcohol, and reflux gently 
on hot plate 1.5 hr. Let cool, and rinse condenser and tip with 
25 mL alcohol. Quant, transfer soln to 400 mL beaker, using 50 
mL alcohol and 100 mL H 2 0. Rinse erlenmeyer with addnl 
portions H 2 to total vol. of 250 mL. 

Add 10 drops thymol blue indicator soln to beaker and, with 



AOAC Methods (1980) 



Heptachlor 



105 



stirring, add HN0 3 (1 +1) dropwise to first pink color of indicator. 
Adjust pH to 2±0.2, using NH 4 OH (1+4) and/or HN0 3 (1+4). 
Adjust total vol. to 300 ml_ with H 2 0. 



6.288 



Determination 



Place sample beaker on mag. stirrer, and adjust to rapid 
stirring. Titr. with AgN0 3 std soln to same mv end point used 
for stdzg AgN0 3 std soln. Titr, blank (unhydrolyzed sample). 

% Active ingredient in tech. Kelthane = 

WJSJ - (V b /S b )] x N x l/ t x 0.1235 x 100}/l/; 

% Active ingredient in Kelthane formulations = 

l(VJS a ) - (V b /S b )] x N x 0.1235 x 100, 
where V s and V b = mL AgN0 3 std soln required to titr. sample 
and blank, resp.; N - normality of AgN0 3 std soln; S a and S b 
= g sample taken for hydrolysis and blank, resp.; V t = total vol. 
sample soln = 500 mL; and V - aliquot vol. sample soln = 25 
mL 

Fluometuron {1,1-Dimethyl-3-(a,a,a-trifluoro-/??-tolyS)urea) 

Gas Chromatographic Method (75) — Official Final Action 

6.289 Standard Solutions 

(a) Diethyl phthalate internal std soln. — Weigh 1.5±0.1 g tech. 
diethyl phthalate, dissolve in ca 100 mL alcohol-free CHCI 3 , dil. 
to 250.0 mL with CHCI 3/ and mix well. Std should be >98% pure 
and contain no impurities eluting at retention time of fluome- 
turon. 

(b) Fluometuron std soln. — Accurately weigh ca 125 mg tech. 
fluometuron of known purity (available from Ciba-Geigy Corp., 
PO Box 11422, Greensboro, NC 27409) into 2 oz round bottle 
with Teflon-lined or Poly-Seal screw cap. Pipet in 25 mL diethyl 
phthalate internal std soln and shake to dissolve. Pipet in 3 mL 
trifluoroacetic anhydride and shake mech. 15 min; then place 
bottle in 55° H 2 bath 30 min. Let cool to room temp. 



6.290 



Preparation of Sample 



Accurately weigh sample contg ca 125 mg fluometuron into 
2 oz round bottle with Teflon-lined or Poly-Seal screw cap. Pipet 
in 25 mL diethyl phthalate internal std soln and shake well. Pipet 
in 3 mL trifluoroacetic anhydride and shake mech. 15 min; then 
place bottle in 55° H 2 bath 30 min. Let cool to room temp. Let 
insol. materials settle or centrf. portion of ext to obtain clear 
soln. 

6.291 Gas Chromatography 

Use instrument equipped with flame ionization detector and 
1.83 m x 2 (id) mm glass column packed with 2% OV-3 (Applied 
Science Laboratories, Inc.) on 80-100 mesh Gas-Chrom Q. 
Condition 24 hr at 240° with N or He at ca 40 mL/min. Column 
should have s=1500 theoretical plates. Use on-column injection 
to prevent decomposition of derivative. 

Typical operating conditions: temps (°) — inlet 150, column 
115±10, detector 250; N or He carrier gas, 20-22 mL/min; air 
and H as specified by manufacturer; attenuation varied so that 
peak hts of pesticide and internal std are 60-80% full scale. 
Retention times for fluometuron derivative and diethyl phthalate 
are 3-5 and 8-10 min, resp. 

6.292 Determination 

Proceed as in 6.433-6.434, except inject 1 fxL aliquots. 



Folpet (A/-(Trichloromethylthio)phthalimide) 

High Pressure Liquid Chromatographic Method (76) 
AOAC-CIPAC Method— Official Final Action 

(Applicable to dry formulations contg folpet as only active 

ingredient and to folpet combination formulations except those 

contg propargite or Me parathion. Compds insol. in CH 2 CI 2 , e.g., 

maneb or inorg. salts, do not interfere.) 



6.294 



Apparatus 



(a) Liquid chromatograph. — Equipped with 254 nm UV detec- 
tor. Typical operating conditions: chart speed, 0.2"/min; eluant 
flow rate, 2 mL/min (ca 800 psi); detector sensitivity, 0.16/4 unit 
full scale; temp., ambient; valve injection vol., 20 /xL. Adjust 
operating conditions to elute folpet peak in 4±1 min. Factors 
such as different H 2 content in CH 2 CI 2 eluant can change 
retention times. Folpet peak must be completely resolved from 
dibutyl phthalate peak which normally elutes in ca 7 min. 

(b) Liquid chromatographic column. — Stainless steel, 300 x 
4 (id) mm, packed with 10 ^m diam. silica gel particles (Waters 
Associates, Inc., No. 27477, or equiv.). 



6.295 



Reagents 



6.293 

See 6.435. 



Calculations 



(a) Eluant — Degassed CH 2 CI 2 . 

(b) Internal std soln. — Accurately weigh ca 0.5 g dibutyl 
phthalate (MC/B Manufacturing Chemists) into 200 mL vol. flask. 
Dil. to vol. with CH 2 CI 2 and mix. 

(c) Folpet std soln.— (100 ju,g folpet + 250 jxg dibutyl phthal- 
ate)/mL. Accurately weigh ca 20 mg folpet ref. std, 99+% pure 
(Chevron Chemical Co., 940 Hensley St, Richmond, CA 94804) 
into glass vial, pipet 20 mL internal std soln into vial, and shake 
to dissolve. Pipet 1 mL into 10 mL vol. flask. Dil. to vol. with 
CH 2 CI 2 . 

6.296 Preparation of Sample 

Accurately weigh sample contg 20 mg folpet into vial. Pipet 
20 mL internal std soln into vial and shake 30 min. Centrf. to ppt 
solids. Pipet 1 mL supernate into 10 mL vol. flask, dil. to vol. 
with CH 2 CI 2 , and mix. Sample contains ca (100 fig folpet + 250 
/Ltg dibutyl phthalate)/mL. 

6.297 Determination 

Inject 20 jul folpet std soln onto column thru sampling valve 
and adjust operating conditions to give largest possible on-scale 
peaks with retention time of 4±1 min for folpet. Repeat injections 
until ratio of folpet to dibutyl phthalate peak hts is within ±1% 
of previous injection. Without changing conditions, inject sample 
soln until its ratio is within ±1% of previous ratio for sample. 
Average last 2 peak ht ratios for sample and for std, resp., and 
calc. % folpet. 

% Folpet = (/?//?') x (W'/W) x P, 
where R and /?' = av. peak ht ratios for sample and std, resp,; 
W = mg folpet in std soln (ca 20 mg); W = mg sample extd for 
analysis; and P ~ % purity of std. 

Heptachlor — Official Final Action 
Active Chlorine Method (61) 

6.298 Reagents 

(a) Dilute acetic acid. — 80%. Dil. 800 mL HO Ac to 1 L with 
H 2 0. 

(b) Silver nitrate-acetic acid std soln. — Dissolve 17 g AgN0 3 
in 200 mL H 2 0, add 56 mL HN0 3 (1 + 1), and dil. to 1 L with 
HOAc. Stdze potentiometrically by adding 25 mL of this soln to 



106 



6. Pesticide Formulations 



AOAC Methods (1980) 



600 mL beaker contg 250 mL 80% HOAc. Immerse glass and Ag 
electrodes in soln and stir with mag. stirrer. Titr. with 0.1/V NaCI 
soln, 6.220(a), to end point (max. change in mv/mL NaCI soln). 
Normality AgN0 3 = mL NaCI x normality NaCI/mL AgN0 3 . 



6.299 



Preparation of Sample 



(a) Emulsifiable concentrate formulations. — Accurately weigh 
sample contg 0.3±0.05 g heptachlor in 250 mL erlenmeyer. 
Dissolve in 50 mL HOAc, and pipet in 25 mL 0.1/V AgN0 3 , (b). 
Attach reflux condenser and reflux 1 hr. 

(b) Granular and dust formulations. — {Caution: See 51.039, 
51.041, and 51.074.) Accurately weigh sample contg 0.3±0.05 g 
heptachlor into 80 x 25 mm Soxhlet extn thimble. Ext 2 hr with 
pentane and transfer ext to 250 mL erlenmeyer. Attach short 
reflux column such as 3-ball Snyder or 12" (30 cm) Vigreux to 
flask and evap. to dryness on steam bath. (Results will be low 
if reflux column is not used.) Rinse down column with 50 mL 
HOAc, pipet in 25 mL 0.1/V AgN0 3 , (b), attach reflux condenser, 
and reflux 1 hr. 

(c) Technical.— Accurately weigh 0.40±0.05 g heptachlor and 
proceed as in (a). 



6.300 



Determination 



Rinse tip of condenser or column with H 2 and cool soln to 
room temp. Transfer quant, to 600 mL beaker, rinsing with four 
10 mL portions 80% HOAc. Immerse glass and Ag electrodes in 
soln and stir with mag. stirrer. Titr. with 0.1/V NaCI soln, 6.220(a), 
to end point. 

% Heptachlor = 37.33 x (25 x normality AgN0 3 soln - mL 
NaCI soln x normality NaCI soln)/g sample. 

Gas Chromatographic Method (77) 
6.301 Apparatus 

(a) Gas chromatography — Equipped with H flame ionization 
detector; capable of accepting glass column and glass-lined 
sample introduction system or on-column injection. Use follow- 
ing conditions: Temps (°): column 175, detector 175-190, sample 
inlet 190; N carrier gas pressure 30 psig; recorder chart speed 
2.5 cm/min. 

(b) Glass-stoppered tubes. — Approx. 25 and 75 mL capacity. 

(c) Microliter syringe. — 10 AtL, Hamilton Co., 701-N. 



6.302 



Reagents 



(a) Heptachlor.— Ret grade (Velsicol Chemical Corp.). 

(b) Aldrin. — Ref. grade (Velsicol Chemical Corp.). 



6.303 



Preparation of Column 



To 9.5 g 100-120 mesh Gas Chrom Q in vac. flask add 0.50 g 
silicone GE Versilube F-50 (available from Applied Science Labs) 
dissolved in 50 mL CH 2 CI 2 . Shake slurry well to wet solid thoroly. 
Connect flask to H 2 aspirator and evap. solv. with frequent 
shaking. When solids appear dry, complete drying by placing 
flask in steam bath and connecting to vac. pump until ca 4 mm 
pressure is attained. Remove flask from steam bath and let cool 
under vac. 

Fill 1.5 m (5') x %" od (0.067" id) Pyrex glass tube with this 
packing, using vac. pump and gentle tapping. Plug ends of 
column with glass wool. Condition column 24 hr in 190° oven 
while purging with N. Let column cool while still purging with 
N; then install in chromatograph. 

6.304 Preparation of Sample 

(a) Liquids. — Weigh sample contg ca 750 mg heptachlor into 



75 mL g-s vial and add 500 mg ref. grade aldrin, Add 75 mL 
fresh CS 2 , stopper, and shake vigorously 2 min. 

(b) Solids. — Transfer weighed sample contg ca 750 mg hep- 
tachlor to Soxhlet and ext 2 hr with 75 mL pentane. Let cool, 
add 500 mg ref. grade aldrin to soln, and swirl. 



6.305 



Calibration 



Weigh 0.2500 g ref. grade heptachlor and 0.1670 g ref. grade 
aldrin into 25 mL g-s flask. Dissolve in 25 mL CS 2 . Chromatograph 
this soln under conditions given in 6.301(a) 5 times to obtain 
accurate response correction factor. (On new column, it is 
sometimes desirable to inject several 5 txL aliquots of std soln 
to condition column before use.) 



6.306 



Determination 



Let instrument equilibrate as in 6.301(a). Inject ca 1 fit sample 
soln at sensitivity setting such that ht of heptachlor peak is ca 
% full scale. For each analysis, allow 10-12 min for heptachlor 
related components to elute. Components and approx. retention 
times in min are: heptachlor 4.5, aldrin 5.9, chlordene 3.1, and 
-y-chlordane 9.9. 



6.307 



Calculations 



Calc. area of heptachlor and aldrin peaks by multiplying peak 
ht in mm by width of peak at half ht in mm. Alternatively, use 
integrator. Calc. response correction factor {f, ca 0.82) for each 
of the 5 std injections as follows: 

f - (area of heptachlor peak x mg aldrin x purity of 
aldrin)/(area of aldrin peak x mg heptachlor x purity of hep- 
tachlor). 

Average 5 replicates and use av. to calc. % heptachlor in 
samples. 

% Heptachlor - (area of heptachlor peak x mg aldrin x purity 
of aldrin x 100)/(area of aldrin peak x mg sample x /). 

Picloram (4-Amino-3,5,6-TrichloropicoHnsc Acid) 
and 2,4-D (2,4-Dichlorophenoxyacetic Acid) 

High Pressure Liquid Chromatographic Method [78) 
Official Final Action 



6.308 



Apparatus 



(a) Liquid chromatograph. — Equipped with 280 nm UV detec- 
tor and injection valve. Alternatively, septum injection head may 
be used; however, stop-flow injection is recommended. Oper- 
ating conditions: eluant flow rate, 0.7 mL/min (ca 1000 psi); 
detector sensitivity, 0.08 A unit full scale; temp., ambient, but 
within ±2.5°. 

(b) Liquid chromatographic column.— No. 316 stainless steel, 
1000 x 2.1 mm id, with Varian No. 96-000075-00 reducing union 
(%" x 1 /i6") contg 2 /x,m frit (regular reducing union packed with 
glass wool may be used instead) packed with DuPont No. 
820960005 Zipax® SAX (strong anion exchange) resin. Preclean 
column with few mL each of CHCI 3 , acetone, and MeOH, and 
vac-dry. Pack in small increments over 40 min period while 
tapping column on hard surface. 

6.309 Reagents 

(a) Eluants.— Prep. sep. solns of 0.0 W Na 2 B 4 O 7 .10H 2 O (3.8 
g/L) and 0.002/W NaCI0 4 .H 2 (0.28 g/L) in previously boiled and 
cooled deionized H 2 0. 

(b) Salicylic acid internal std soln. — Accurately weigh ca 3.6 
g USP Ref. Std Salicylic Acid into 1 L vol. flask, dil. to vol. with 
0.05/V NaOH in isopropanol-HX* (1+1), and mix. 



AOAC Methods (1980) 



Amitrole 



107 



(c) Picloram-2,4-D std sofn. — (4 mg picloram + 12 mg 2,4-D 
+ 3.6 mg salicylic acid)/mL. Accurately weigh ca 100 mg 
picloram ref. std, 99 + % pure (Dow Chemical Co.), and ca 300 
mg 2,4-D ref. std, 99 + % pure (Dow Chemical Co.), into glass 
vial, pipet in 25 mL salicylic acid internal std soln, and shake to 
dissolve. 



6.310 



Preparation of Sample 



Accurately weigh ca 1.6 g sample into ca 10 dram glass vial, 
pipet in 25 mL salicyclic acid internal std soln, and shake to 
dissolve. 



6.311 



Determination 



Inject 2 fxL picloram-2,4-D std soln onto column and adjust 
attenuation to give largest possible on-scale peaks. Repeat 
injections until peak ht ratios of herbicide: internal std vary ^1% 
for successive injections. Without changing conditions, inject 2 
jjlL aliquots sample soln until peak ht ratios vary ^1%. Average 
last 2 peak ht ratios for picloram and 2,4-D and calc. % herbicide. 

% Herbicide = (RJR S ) x (W s /W x ) x P, 
where R K and R s = av. peak ht ratios of each herbicide to the 
internal std for sample and std, resp.; W s - mg herbicide in std; 
W x - mg sample; and P ~ % purity of std. 

Sodium Trichloroacetate (79) — Official Final Action 

{Caution: See 51.011, 51.039, 51.041, and 51.070.) 
6.3 1 2 Apparatus and Reagent 

(a) Reflux apparatus. — 250 mL erlenmeyer attached thru % 
24/40 joint to 50 cm water-cooled condenser. 

(b) Dioxane. — Freshly distd. 



6.313 



Determination 



Dissolve 25 g sample in H 2 and dil. to 100.0 mL. Pipet aliquot 
(usually 10 mL), titrg ca half that of blank, into 250 mL refluxing 
flask, add 1 drop Me red, and neutze with ca 1/V H 2 S0 4 to distinct 
orange-pink. pH is 5.3-5.5; usually <0.15 mL is required. If soln 
is acid, titr. with ca 1/V NaOH. Add 25.00 mL 1/V H 2 S0 4 , 35 mL 
dioxane, and few glass beads. Boil vigorously under reflux 2=60 
min. Cool, add 2 drops Me red, and titr. with std W NaOH to 
sharp change from orange to yellow end point. Perform blank 
detn, omitting sample. 

% Na trichloroacetate = Net mL 1/V acid x 0.1854 x 100/g 
sample in aliquot. 

6.314 Trifluralin — Official First Action 
See 6.203-6.214. 



NONHALOGENATED PESTICIDES 

Aldicarb (2-Methyl-2-(methylthio)propionaldehyde-0- 
(methylcarbamoyl) Oxime) (80)— Official Final Action 

{Caution: See 51.041.) 
6.3 1 5 Apparatus and Reagents 

(a) Infrared spectrophotometer. — Perkin-Elmer Model 337, or 
equiv. Adjust conditions as required by specific instrument. 

(b) Sox h let extractor. — With 125 mL flask and 25 x 80 mm 
cellulose thimble. 



(c) Aldicarb std soln. — 0.18 g/100 mL. Accurately weigh (to 
0.1 mg) 0.18+0.01 g anal, grade aldicarb (available from Union 
Carbide Corp., Agricultural Products and Services) into 100 mL 
g-s vol. flask, add ca 80 mL CH 2 CI 2 , mix to dissolve, and dil. to 
vol. with CH 2 CI 2 . 



6.316 



Determination 



Transfer accurately weighed sample contg 0.18±0.01 g aldi- 
carb to extn thimble, cover with wad of surgical grade cotton, 
and place thimble in extractor. Add 2-3 Alundum boiling stones 
and ca 80 mL CH 2 CI 2 to flask, and ext at rate to provide 5 extns 
within 60 min. Let cool to room temp., transfer quant, to 100 mL 
g-s vol. flask with CH 2 CI 2 , and dil. to vol. 

Using matched 0.5 mm NaCI cells, scan sample and std solns 
from 5.2 to 6.0 fim (1900 to 1600 crrr 1 ) against CH 2 CI 2 . Calc. A 
of sample and A' of std at 5.75 ptm (1740 cm -1 ), using corre- 
sponding A at 5.4 [inn (1850 crrr 1 ) as / . {A and A' should both 
be ca 0.45.) 



6.317 



Determination of Binder Correction 



Pipet 50 mL sample soln into 100 mL beaker and place in 
room temp. H 2 bath in hood. Evap. to dryness, using gentle 
stream of clean, dry air. Add 25 mL MeOH, stir well, and filter 
thru 30 mL coarse fritted glass gooch. Rinse beaker and gooch 
with 25 mL MeOH, applying vac. until all liq. is in filter flask. 
Place gooch and contents in original beaker, place 20 mL CH 2 CI 2 
in gooch, and swirl to dissolve binder, letting solv. drip into 
beaker. Repeat with addnl 20 mL CH 2 CI 2 . Quant, transfer solv. 
to 50 mL g-s vol. flask and dil. to vol. with CH 2 CI 2 . Scan soln as 
in detn and subtract A of binder soln (should be <0.005) from 
that of sample (=A>4). 

% Aldicarb by wt = (A>4/g sample) x (g std/>V) x P, where 
P is % purity of ref. std. 



Amitrole (3-Amino-s-triazole) (81) — Official Final Action 



{Caution: See 51.018 and 51.041.) 



6.318 



Preparation of Sample Solution 

(a) 50% Dry powder formulation.— -Transfer 10.00 g sample 
to 100 mL g-s vol. flask, using powder funnel. Add 50 mL DMF. 
Shake 2-3 min to dissolve amitrole. (Undissolved amitrole is 
powder and can be differentiated visually from inerts which are 
usually crystals.) Let settle and carefully decant supernate into 
100 mL vol. flask. Repeat extn of residue with three 15 mL 
portions DMF, letting settle each time before decanting into vol. 
flask. Dil. combined exts to vol. with DMF and shake well. Filter 
40-50 mL thru fritted glass filter of medium porosity. Pipet 25 
mL into 400 mL beaker contg 50 mL H 2 0. 

(b) 90% Dry powder formulation.— Dissolve 1.0000 g sample 
in 100 mL H 2 in 400 mL beaker. 

(c) Aqueous amitrole. — Pipet 5 mL sample into 400 mL beaker 
contg 50 mL H 2 0. 



6.319 



Determination 



Adjust sample soln or dild aliquot to pH 1.8 with 0.5/V HCI. 
Stir mech. and titr. with 0.5 mL increments 0.5/V NaOH to pH 
3.5-4.0. (Use Beckman Model G pH meter, or equiv., equipped 
with glass-calomel electrode system, and stdzd at pH 4.0 and 
7.0 with buffers, 50.007(c) and (d).) Add 0.5/V NaOH rapidly to 
pH 6.5 and then dropwise to pH 7.5 (second inflection point). 
Plot pH against mL 0.5/V NaOH and det. first inflection point 
(occurs at pH 2.5-2.9). 



108 



6. Pesticide Formulations 



AOAC Methods (1980) 



% Amitrole by wt = [B - C) x 0.5 x 8.408/f , 
where C - mL 0.5/V NaOH required to titr. to first inflection 
point; B = mL 0.5/V NaOH required to titr. to pH 7.5; and F = 2.5 
for 50% dry powder formulation, (a), g sample for 90% dry 
powder formulation, (b), and 5.0 x sp gr sample for aq. amitrole, 
<c). 

lb Amitrole in aq. amitrole/U.S. gal. = % amitrole x sp gr x 
8.32/100. 

Carbaryl (1-Naphthyl Methylcarbamate) (82) 
Official Final Action 

{Caution: See 51.018, 51.040, 51.041, and 51.056.) 

6.320 Apparatus 

(a) Centrifuge. — Clinical model, 8 place, or equiv. 

(b) Hypodermic syringe. — 1 mL, glass barrel with rubber- 
tipped plastic plunger (1 mL B-D Glaspak Tuberculin disposable 
syringe supplied by Becton, Dickinson, and Co. is suitable). 
Disposable syringe may be used repeatedly. Wash with H 2 and 
acetone or MeOH, air-dry, and lubricate rubber plunger tip with 
silicone stopcock grease. 

(c) Infrared spectrophotometer. — Perkin-Elmer Corp., Model 
337, or equiv. Operator must adapt conditions to instrument. 

<d) Rotator. — Tube type, BBL, or equiv. 

.(e) Shaking machine. — Wrist-action shaker (Burrell Corp., or 
equiv.). 

(f) Tubes. — Culture tubes, borosilicate glass, 16 x 150 mm 
with screw caps and Teflon liners (Corning Glass Works No. 
9826, or equiv.). 



6.321 



Reagents 



(a) Methanol-chloroform sofn.—W% (v/v) MeOH in CHCI 3 . 

(b) Carbaryl std solns.— {1)8 mg/mL — Transfer 0.12±0.01 g 
carbaryl (anal, grade, available from Union Carbide Corp., 
Agricultural Products and Services), weighed to nearest 0.1 mg, 
to culture tube. Pipet 15 mL MeOH-CHCI 3 soln into tube, cap 
securely, and rotate or shake mech. 30 min. {2) 2.5 mg/mL — 
Transfer 0.25±0.01 g carbaryl, weighed to nearest 0.1 mg, to 
250 mL g-s erlenmeyer. Pipet 100 mL CHCI 3 into flask, stopper, 
and swirl to dissolve. 

6.322 Preparation of Sample 

(a) Carbaryl dust and powder formulations. — Transfer 
weighed sample (^2.4 g) contg 0.12±0.01 g carbaryl to culture 
tube. Pipet 15 mL MeOH-CHCI 3 soln into tube and cap securely. 
Rotate or shake mech. 30 min and centrf. 10 min. 

(b) Liquid suspensions. — Following steps must be performed 
in order described, as any deviation can cause erroneous results 
due to faulty sample transfer and incomplete extn: Place ca 20 
g IMa 2 S0 4 in 250 mL g-s erlenmeyer. Pipet 100 mL CHCI 3 into 
flask. Vigorously shake sample bottle. Draw appropriate vol. 
sample into hypodermic syringe without needle. Use ca 0.5 mL 
sample for carbaryl 4 lb/gal. and ca 1 .0 mL for carbaryl 2 lb/gal. 
Wipe outside of syringe with paper towel and weigh syringe 
and contents to nearest 0.1 mg. Add sample to erlenmeyer by 
slowly depressing syringe plunger. Do not let syringe or sample 
touch sides of flask. Sample must drop into CHCI 3 . Reweigh 
syringe and calc. sample wt by difference. Stopper flask and 
shake vigorously 30 min on mech. shaker. 



6.323 



Determination 



Measure A of carbaryl peak at 5.75 /aiti (1740 crrr 1 ), using A at 
5.40 /Ltm (1850 cm" 1 ) as point. A = ca 0.4 for both std and 
sample. 

% Carbaryl by wt = {A x B' x P)/{A' x B), 
where A and A ' = absorbance of sample and std, resp., at 5.75 
/i,m; B and B' = mg sample and mg std/mL, resp.; and P = % 
purity of carbaryl std. 

(b) Liquid suspensions, — Proceed as in (a), except use 
matched 0.5 mm NaCI cells and scan sample soln against CHCI 3 . 



2,2-DachlorovmyS Dimethyl Phosphate 
(DDVP) (83)— Official First Action 

Method I 

(Applicable to sand/sugar base fly bait contg ca 0.5% and 4 
lb/gal. DDVP emulsifiable cones. Caution: See 51.041.) 

6.324 Apparatus and Reagent 

(a) Infrared spectrophotometer. — Capable of recording in re- 
gion 2-15 fim. Slit width must be adjustable to give signal-to- 
noise ratio of ca 100:1; with sealed liq. absorption cell, NaCI 
windows, and 0.2 mm path length. 

.(b) Hypodermic syringe. — Luer type, glass, 1.0 mL. Use 18 
gage (Stubbs), 2" slip-on needle. 

(c) 2,2-Dichforovinyf dimethyl phosphate. — Use std DDVP of 
known purity. {Available from Shell Chemical Co.) 

6.325 Calibration of Apparatus 

Into each of five 10 mL vol. flasks, weigh, to nearest 0.1 mg, 
25, 75, 100, 150, and 200 mg DDVP std, and dil. to vol. with 
CHCI 3 . Calibration solns contain ca 2.5, 7.5, 10, 15, and 20 g 
DDVP/L. 

Fill sealed liq. absorption cell with CHCI 3 , adjust spectrophtr 
to optimum settings, and scan over 10.7-9.9 jum. Without 
changing settings, fill cell in turn with each of prepd calibration 
solns, starting with most dil., and scan each soln over 10.7-9.9 
/im. 

For each scan, construct baseline thru absorption min. at ca 
10.0 fjim parallel to radiation line. Draw perpendicular to 
radiation line thru absorption max. of calibration soln at ca 10.2 
/xm and measure radiant power P (at 10.0 ^m) and P (at 10.2 
ju,m), in any convenient units but keeping same units thruout. 
Calc. A as log (P /P). Repeat calcns, using absorption min. at ca 
10.5 fjum as ref. point. 

Subtract A of cell and CHCI 3 obtained above from A of cell 
and calibration solns. Plot AA of DDVP as ordinate against g/L 
DDVP as abscissa for each ref. point (10.0 and 10.5 /Am). 



6.326 



Preparation of Sample Solution 



(a) Carbaryl dust and powder formulations. — Using matched 
0.2 mm NaCI cells, scan sample soln against MeOH-CHCl 3 soln 
from 5.2 to 6.0 /urn (1900-1600 cm" 1 ). Repeat scan with std soln. 



(a) Sand /sugar base fly baits. — Prep. 25 x 400 mm extn 
column by adding enough diat. earth (Hyflo Super-Cel) to make 
layer 5 cm high when gently packed. Place 250 mL vol. flask 
under outlet. Accurately weigh sample contg 0.2-1.0 g DDVP. 
Transfer sample to extn column with CHCI 3 , and rinse sample 
container with CHCI 3 . 

Working in well-ventilated hood, add 50 mL CHCI 3 to column. 
Using stirring device, vigorously agitate sample and top half of 
adsorbent layer to form slurry with solv. Withdraw stirring 
device, and rinse it and column with addnl CHCI 3 from wash 
bottle. Let solv. percolate thru column until level is few mm 
above diat. earth-sample layer. 

Add ca 50 mL CHCI 3 to column, agitate sample and diat. earth 
with stirrer as above, and let solv. percolate thru column until 
upper level approaches sample layer. Repeat with two addnl 50 



AOAC Methods (1980) 



Paraquat 



109 



mL portions CHCI 3 . When solv. ht has diminished to 2-3 mm, 
rinse column with three 10 mL portions CHCI 3 , letting each 
portion enter diat. earth layer before adding next. Let column 
drain and rinse outlet tip with CHCI 3/ collecting rinse in 250 mL 
vol. flask. 

Transfer CHCI 3 eluate to evapg dish (125 mm diam.) marked 
at 40-50 mL. Evap. on steam bath to 4(0-50 mL. Remove dish 
and continue evapn at room temp, to 10-15 mL. Using CHCI 3 , 
quant, transfer to vol. flask of such size to give DDVP concn of 
0.5-1.0 g/100 mL when soln is dild to vol. 

(b) Emulsifiabie concentrates. — Weigh enough sample, to 
nearest 0.2 mg, to give ca 1 g DDVP/100 mL CHCI 3 when dild 
to vol. in 10, 25, or 50 mL vol. flask. 



6.327 



Determination 



Dil. CHCI 3 soln of DDVP to vol. with CHCI 3 , mix thoroly, and 
fill calibrated liq. absorption cell with sample soln. Using same 
instrument settings as for calibration, scan sample soln over 
10.7-9.9 /aid. 

Examine spectra for possible interference and use appropriate 
absorption min. as ref. point. (If solvs or other ingredients 
interfere at one of ref. points, use alternative ref. point.) For 
example, 0-naphthol, often used as stabilizer in fly baits, exts 
with CHCI3 and absorbs at ca 10.5 /xm, requiring use of 10.0 /xm 
ref. point. 

Calc. A of sample soln as in 6.325. 

From calcd/4, read g DDVP/L from calibration curve. 

% DDVP by wt - [(g DDVP/L) x mL sample soln]/ 
(10 x g sample). 



Method II (84) 

(Applicable to ca 0.5% (w/w) spray soln and ca 
1.0% (w/w) cattle spray in hydrocarbon solvs) 



6.328 



Apparatus and Reagent 

Double beam instrument 



(a) Infrared spectrophotometer. 
with specifications as in 6.324(a). 

(b) 2,2-Dichlorovinyl dimethyl phosphate. — See 6.324(c). 

6.329 Preparation of Compensating Solvent 

Transfer ca 30 mL sample to 125 mL separator and ext (2-3 
min per extn) with 4 ca 30 mL portions 0.5/V NaOH. Dry DDVP- 
free hydrocarbon phase by passing it thru 2-3 g anhyd. Na 2 S0 4 . 
Reserve dried solv. for prepn of DDVP std soln and as compen- 
sating solv. in ref. cell. 

6.330 Determination 

Prep, std DDVP soln in compensating solv. that approximates 
(on wt basis) DDVP content of sample. Calc. DDVP content of 
std soln to nearest 0.01% by wt. 

After detg optimum instrument parameters for compensation 
technic, scan std soln over 9.9-10.7 itm (1010-935 cm -1 ) region 
with ref. cell contg compensating solv. in ref. beam of spec- 
trophtr. Scan sample against compensating solv. in same man- 
ner. 

From d ifferential spectra, det. A of DDVP at 1 0.2 /xm (980 cnrr 1 ) 
of std, A', and sample, A, measured from baseline drawn 
between minima near 10.0 and 10.6 ^m. Calc. DDVP as follows: 

% DDVP by wt = % DDVP in std x A/ A'. 

6.331 Diazinon— Official First Action 
See 6.431-6.435. 



Diquat (6,7-Dihydrodipyrido (1,2na:2',1'-c) 
Pyrazinediium ton) (85) — Official Final Action 

AOAC-CIPAC Method 



6.332 



Reagents 



(a) Acetate buffer soln.^pH 4.05. Dissolve 10.88 g NaOAc 
.3H 2 in H 2 0, add 19 mL HOAc, dil. to 2 L with H 2 F and mix. 

(b) Diquat std solns. — (1) Stock soln. — 0.2 mg diquat/mL. 
Prep, stock soln by dissolving 0.1968 g pure diquat dibromide 
monohydrate (C 12 H 12 N 2 Br 2 .H 2 0, MW 362.1 ; 50.87% cation; avail- 
able from Chevron Chemical Co., 940 Hensley St, Richmond, CA 
94804) in buffer soln, dil. to 500 mL with buffer soln, and mix. 
(2) Working soln.— 0.02 mg diquat/mL. Dil. 10.0 mL stock soln 
to 100 mL with buffer soln. Prep, dild stds fresh as required. 



6.333 



Determination 



Using buret, transfer 10.0, 20.0, and 30.0 mL std diquat soln, 
contg 0.2, 0.4, and 0.6 mg diquat, resp., to three 100 mL vol. 
flasks, dil. each soln to vol. with buffer soln, and mix. Measure 
A of stds at 310 nm in 1 cm silica cell, with buffer soln as ref., 
and draw std curve relating /4 to mg diquat. 

Accurately weigh portion (w g) of well mixed sample contg 
ca 0.5 g diquat, transfer to 250 mL vol. flask, dil. to vol. with 
buffer soln, and mix (Soln /). Transfer 10.0 mL Soln 1 to 200 mL 
vol. flask, dil. to vol. with buffer soln, and mix (Soln 2). Transfer 
5.0 mL Soln 2 to 100 mL vol. flask, dil. to vol. with buffer soln, 
and mix {Soln 3). 

Measured of Soln 3 at 310 nm in 1 cm silica cell, with buffer 
soln as ref. Read diquat content of Soln 3 (y mg) directly from 
std curve or calc. diquat content by interpolation. 
% Diquat, w/w = 100 y/w. 



Paraquat (1,1 M3imethyl-4,4'-bipyridinium 
Ion) (85)— Official Final Action 



6.334 



Reagents 



(a) Sodium dithionite.—IYo soln in 0.1/V NaOH. (Sodium 
dithionite, Na 2 S 2 4 .2H 2 0, is also called sodium hydrosulfite and 
sodium hyposulfite.) Do not keep soln >3 hr; solid is unstable 
in presence of moisture. Store solid in small air-tight bottles in 
vac. desiccator. 

(b) Paraquat std soln. — 0.25 mg paraquat/mL. Dry anal, std 
(available from Chevron Chemical Co., 940 Hensley St, Rich- 
mond, CA 94804) to const wt at 100-120° before weighing 
(paraquat salts are hygroscopic). Dissolve 0.1728 g paraquat 
dichloride (72.40% cation) in H 2 0, dil. to 500 mL with H 2 0, and 
mix. Prep, soln fresh as required. 

(c) Extracting soln. — Dissolve 11 g Na 2 SO 4 .10H 2 O in 500 mL 
H 2 0, add 500 mL alcohol, and mix. 

6.335 Preparation of Standard Curve 

Pipet 50 mL std soln into 250 mL vol. flask, dil. to vol. with 
H 2 0, and mix. Pipet 5, 10, 15, and 20 mL aliquots of this dild std 
soln into sep. 100 mL vol. flasks. (When dild to vol. these solns 
contain 2.5, 5.0, 7.5, and 10.0 fxg paraquat/mL, resp.) Proceed 
as in 6.337. Plot/4 against jug paraquat/mL at final diln. 

6.336 Preparation of Sample 

(Caution: Open aerosol can behind safety shield.) 

(a) Formulations not containing oil base. — Accurately weigh 
portion well mixed sample contg ca 0.25 g paraquat. Transfer 
to 500 mL vol. flask, dil. to vol. with H 2 0, and mix well (Soln /). 
Pipet 10 mL Soln 1 into 100 mL vol. flask, dil. to vol. with H 2 0, 



110 



6. Pesticide Formulations 



AOAC Methods (1980) 



and mix well [Soln 2). Pipet 10 ml_ Soln 2 into 100 mL vol. flask 
and proceed as in 6.337. 

(b) Aerosol formulations containing oil base. — Weigh aerosol 
can to nearest 0.1 g (C). Clamp can with bottom up and puncture 
smallest possible hole with punch and hammer. After hiss of 
escaping propellent is no longer heard, cut bottom Ve open with 
hand can opener. Push nearly detached lid into can. Immerse 
can 15 min in 50-70° H 2 bath or in hot tap H 2 running into 1 
L beaker. 

Add 50 mL extg soln, (c), and 50 mL pentane to 250 mL 
separator. Remove can from H 2 bath, dry well (especially inside 
cap and around valve), and weigh (O). Place pipet with capacity 
to deliver ca 20 mg paraquat in can, and weigh both (£). 
Withdraw liq., transfer contents to separator, replace pipet in 
can, and weigh (F). (Disregard material left in and on pipet.) 
Empty can, rinse completely with acetone, air dry, and weigh 
(G). 

Stopper separator and shake 30 sec, venting frequently. Let 
layers sep, and drain lower layer into 200 mL vol. flask. Add 25 
mL extg soln to separator, repeat extn, and drain lower layer 
into same vol. flask. Dil. to vol. with extg soln and mix well. 
Pipet 5 mL into 100 mL vol. flask and proceed as in 6.337. 



6.337 



Determination 



(Complete analysis of one soln before adding 
dithionite to next soln.) 



Add 10 mL Na dithionite soln to one 100 mL vol. flask and dil. 
to vol. with H 2 0. Mix by inverting end-over-end 3 times at such 
speed that air bubble travels from one end to other; do not 
shake flask vigorously, as this tends to cause fading of color 
due to oxidn. Immediately measured of soln at 600 nm, using 
reagent blank (no paraquat) to set the 100% T or for ref. side for 
dual beam instruments. Similarly, treat each flask in turn, 
completing color measurement without delay before adding 
dithionite to next soln. 

% Paraquat = (jug/mL from std curve) x 5/g sample. 

% Paraquat (in aerosol formulations) = [(/-ig/mL from std 
curve) x [D - G) x 0.4]/[(C - G) x (£ - F)]. 



Dithiocarbamates (Ferbam, Ma neb, 
Nabam, Zineb, and Ziram) 

{Caution: See 51.041.) 

Carbon Disulfide Evolution Method [86) 
Official Final Action 

(Applicable only to cones or formulations 
free from interfering substances) 



6.338 



Principle 



Dithiocarbamates decompose on heating in acid medium. 
Evolved CS 2 is passed thru Pb(OAc) 2 soln traps to remove H 2 S 
and S0 2 formed from sample impurities. Washed CS 2 is reacted 
with methanolic KOH, and xanthate formed is titrd with I soln. 

6.339 Apparatus 

Carbon disulfide evolution apparatus. — See Fig. 6:07. Avail- 
able from Scientific Glass Apparatus Co., No. JE-1000. 

6.340 Reagent 

Methanolic potassium hydroxide. — 2/V. Dissolve 112 g KOH 
pellets in 500 mL anhyd. MeOH, filter thru cotton, and add addnl 
500 mL anhyd. MeOH, 



57 MM 01k BULBS 

WITH 4 INDENTATIONS 

EACH 



KOH-MEOH ABSORBER 



HEAVY WALL TUBING 



I $ SPHERICAL JOINT 



20 MM TUBING - 



LEAD ACETATE TRAPS 




REACTION FLASK 
(2NECK-250HL) 



FIG. 6:07 — Carbon disulfide evolution apparatus 



6.341 



Determination 



Add 20 mL 10% Pb(OAc) 2 soln to each Pb(OAc) 2 trap and pipet 
50 mL IN MeOH-KOH soln into MeOH-KOH absorber (Fig. 
6:07). (Absorber must be dry at time of addn and kept at 25±1°.) 
Add 50 mL H 2 S0 4 (1+4) to reaction flask and heat acid to boiling. 
Adjust aspiration rate to =^1 bubble/ sec thru MeOH-KOH soln, 
using stopper in reaction flask. 

Weigh ^5 g sample (contg 0.1-0.3 g dithiocarbamates) into 
small filter paper cone and fold cone to prevent sample loss. 
Remove stopper from reaction flask, insert wrapped sample, 
and immediately stopper flask. Adjust air flow if necessary and 
maintain steady, moderate boil. Do not let acid soln enter air 
inlet tube. Some dust formulations react vigorously and require 
special care to prevent ejection of hot acid. As reaction proceeds, 
adjust system so that rates of boiling and aspiration are almost 
in equilibrium, producing only very slow rate of bubbling thru 
MeOH-KOH soln. Continue boiling 1.5 hr. Disconnect MeOH- 
KOH absorber and rinse contents into 500 mL erlenmeyer, using 
ca 250 mL H 2 0. (To remove absorber contents, apply slight air 
pressure to top of absorber and force soln thru side arm. Rinse 
with 4 ca 25 mL portions H 2 0, forcing out rinse H 2 in same 
manner with air pressure.) 

Add 3 drops phthln, and titr. with 30% HOAc until red just 
disappears. Immediately titr. with 0.1/V I; near end point, add 5 
mL starch indicator soln, 6.005(f), and titr. to faint but definite 
color change. 

Det. blank (usually 0.1-0.2 mL 0.1/V I) by dilg 50 mL MeOH- 
KOH soln with 250 mL H 2 0, neutzg with 30% HOAc, and titrg as 
above. 

Calc. % dithiocarbamate = (Sample titrn - blank) x (I nor- 
mality) x (equiv. wt dithiocarbamate)/(g sample x 10). 

Equiv. wts ( 1 / 2 MW) of zineb, maneb, ziram, nabam, and (Y 3 
MW) ferbam are 137.87, 132.65, 152.91,128.18, and 138.82, resp. 



AOAC Methods (1980) 



Formaldehyde 



111 



Thiram (Bis(dimethylthiocarbamoyl)disulfide) 
(Tetramethylthiuram Disulfide) 

CIPAC Method [87)— Official Final Action 

6.342 Principle 

Thiram is decomposed by boiling with HOAc and Zn(OAc) 2 to 
Me 2 NH, CS 2 , and carbonyl sulfide. The gaseous mixt. is carried 
by air stream thru CdS0 4 scrubber to remove H 2 S, and then into 
absorption system contg MeOH-KOH soln. Mixed xanthate- 
monothiocarbamate soln is neutzd and titrd with std aq. I. 

Method is not specific for thiram. Sep. characterization test, 
29.171, must be made. 



6.343 



Apparatus 



Assembly and operating conditions. — Assemble a pp. as 
shown in Fig. 6:08 with 30 mL CdS0 4 soln in first absorber, 25 
ml_ KOH soln in second absorber, and 5 mL in each bubbler. 
Turn on condenser H 2 and maintain H 2 bath surrounding 
CdS0 4 scrubber at 70-80° thruout test. Keep main KOH absorber 
at <25° by immersion in beaker of cold H 2 0. Absorber must be 
dry or rinsed with MeOH before adding KOH soln. Air bleed 
must reach nearly to bottom of digestion flask. Make all joints 
gas-tight, using small amtsH 3 P0 4 , petrolatum, or silicone grease. 

Check app. for absorber leaks and efficiency periodically, 
using pure Na diethyldithiocarbamate. Recoveries should be 
99-101%. Check purity of Na diethyldithiocarbamate by dis- 
solving ca 0.5 g, accurately weighed, in 100 mL H 2 and titrg 
directly with 0.1/V I, using ca 2% starch soln as indicator. 1 mL 
0.1/V I = 0.02253 g Na diethyldithiocarbamate. % Na diethyldi- 
thiocarbamate = 2.253 x mL 0.1/V l/g sample. 

6.344 Reagents 

(a) Acid mixture.— Dissolve 2.5 g ZnO in 100 mL HOAc (1 + 1). 

(b) Cadmium sulfate soln. — Dissolve 18.5 g 3CdS0 4 .8H 2 in 
100 mLH 2 0. 

(c) Potassium hydroxide soln.—2N in MeOH and contg <1 
ppm Cu or Fe. 

(d) Iodine std soln. — 0.1/V. Stdze as in 50.019. 



6.345 



Determination 



Accurately weigh and transfer sample contg ca 0.3 g thiram 
to digestion flask, using small amt H 2 0, if necessary- Assemble 
air bleed and dropping funnel, Fig. 6:08, and add 20 mL acid 
mixt. thru funnel. Connect app. to controlled aspiration (vac. or 
compressed air) so that ca 3 bubbles/sec pass thru absorbers. 
After sample is evenly dispersed, heat and reflux 30 min at 
moderate rate. Turn off cooling H 2 and flush condenser and 
first absorber with steam from flask ^1 min. Remove burner 



and disconnect train. Wash contents of KOH absorber and 
bubblers into 600 mL beaker with 300-400 mL H 2 0, add 1-2 
drops phthln, just neutze with HOAc (1+9) from buret, and add 
3 drops excess. With continual stirring, titr. immediately (pref- 
erably within 1 min, as decomposition of mixed xanthate/ 
monothiocarbamate soln is extremely rapid under acidic con- 
ditions) with 0.1/V I {t mL), using ca 2% starch soln as indicator. 
Det. blank in same manner, omitting sample {b mL). 1 mL 0.1/V 
I = 0.01202 g thiram. 
% Thiram = 1.202 {t - b)/g sample. 

Dodine (n-Dodecylguanidine acetate) [88) 
Official Final Action 

{Caution: See 51.022, 51.028(a) and (d), and 51.041.) 

6.346 Reagents 

(a) Perchloric acid.-0.05N. Dissolve 4.2 mL 72% HCI0 4 in 
HOAc and dil. to 1 L with HOAc. Stdze as follows: Accurately 
weigh 0.200 g KHC e H 4 4 into 250 mL erlenmeyer. Dissolve in 20 
mL HOAc by gently heating flask on hot plate. Add 80 mL Ac 2 
and 8 drops metanil yellow indicator, (b). Place erlenmeyer 
contg bar on mag. stirrer and titr. with HCI0 4 to first definite red 
(magenta). Titr. reagent blank and correct sample titer. 

Normality - 0.200/(0.20422 x net mL HCIOJ 

(b) Metanil yellow.— 0.20%. Dissolve 0.200 g metanil yellow 
powder in 100 mL MeOH. 

(c) Potassium acid phth a late. —NBS SRM KHC 8 H 4 4 . 



6.347 



Determination 



Accurately weigh sample contg ca 0.600 g dodine into 250 mL 
erlenmeyer. Add 10 mL HOAc followed by 90 mL Ac 2 0. Mix by 
swirling 5 min. Filter slurry with vac. thru large, medium porosity 
fritted glass buchner into 250 mL vac. flask. Wash erlenmeyer 
and residue in funnel with two 10 mL portions HOAc-Ac 2 
(10+90). Place vac. flask contg bar on mag. stirrer, add 8 drops 
metanil yellow indicator, and titr. with stdzd ca 0.05/V HCI0 4 to 
first definite red (magenta). Titr. reagent blank and correct 
sample titer. 
% Dodecylguanidine acetate 

= (net mL HCI0 4 x normality x 28.75)/g sample 

Formaldehyde in Solutions — Official Final Action 

Hydrogen Peroxide Method {89) 

6.348 Reagents 

(a) Sulfuric acid std soln. — 1/V. Prep, and stdze as in 
50.039-50.041 



-BIO Join! 



20 ml 



50 ml. 



Shield 




FIG. 6:08 — Absorption system for thiram. Dimensions in cm; N.S. = nonstandard; B10 = $" 10/30 



112 



6. Pesticide Formulations 



AOAC Methods (1980) 



(b) Sodium hydroxide std soln. — 1/V. Stdze against (a), using 
litmus or bromothymol blue indicator. 1 mL = 30.03 mg HCHO. 

(c) Hydrogen peroxide soln. — Com., contg ca 3% H 2 2 - If acid, 
neutze with NaOH, (b), using litmus or bromothymol blue 
indicator. 

(d) Litmus indicator.-— -Soln. of purified litmus of such concn 
that 3 drops gives distinct blue color to 50 mL H 2 0. 

(e) Bromothymol blue indicator. — Dissolve 1 g bromothymol 
blue in 500 mL alcohol, 50% by vol. 



6.349 



Determination 



Pipet 50 mL 1/V NaOH soln into 500 mL erlenmeyer and add 
50 mL H 2 2 , (c). Add weighed amt sample (ca 3 g) from 
weighing pipet, letting point of pipet reach nearly to liq. in flask. 
Place funnel in neck of flask and heat on steam bath 5 min, 
shaking occasionally. Remove from bath, wash funnel with H 2 0, 
cool to room temp., and titr. excess NaOH with std acid, using 
bromothymol blue or litmus. (Cool flask before titrn to obtain 
sharp end point with litmus.) From mL 1/V NaOH used and wt 
sample, calc. % HCHO according to following equation 
NaOH + HCHO + H 2 2 - HCOONa + 2H 2 0. 

If HCHO soln contains appreciable free acid, titr, sep. portion 
and calc. acidity as % HCOOH. Correct for this acidity in calcg 
% HCHO. 

6.350 Cyanide Method (90) 

(Applicable only to dil. solns) 
Treat 15 mL 0.1/V AgN0 3 , 50.027-50.029, with 6 drops HN0 3 
(1 + 1) in 50 mLvol. flask, add 10 ml KCN soln (3.1 g in 500 mL 
H 2 0), dil. to vol., shake well, filter thru dry filter, and titr. 25 mL 
filtrate with 0.1/V NH 4 SCN, 50.003-50.004, as in 3.074. Acidify 
another 15 mL portion 0.1 N AgN0 3 with 6 drops HN0 3 (1+1) 
and treat with 10 mL of the KCN soln to which has been added 
measured amt of sample (wt calcd from sp gr) contg ^25 mg 
HCHO. Dil. to 50 mL, filter, and titr. 25 mL aliquot with the 0.1/V 
NH 4 SCN as before. Difference between mL NH 4 SCN used in 
these 2 titrns x 2 = mL 0.1/V NH 4 SCN corresponding to KCN 
used by the HCHO. Calc. % HCHO present. 1 mL 0.1/V NH 4 SCN 
- 3.003 mg HCHO. 

6.351 Formaldehyde in Seed Disinfectants (91) 
Official Final Action 

(Applicable to detn of HCHO absorbed in inert carrier, e.g., 
bentonite, talc, charcoal, sawdust) 

Weigh ca 5 g sample contg 0.3-0.5 g HCHO in weighing bottle 
and transfer to 800 mL Kjeldahl flask. Add 25 mL H 2 and 12 mL 
H 2 S0 4 (1+4). Steam distil rapidly, passing vapors thru condenser 
with delivery end dipping into 25 mL H 2 in 500 mL vol. flask. 
Collect ca 450 mL distillate, keeping vol. in distg flask nearly 
const with aid of small flame. After distn, wash delivery tube, 
and dil. distillate to vol. with H 2 0. 

Into each of two 200 mL vol. flasks measure 20 mL 0.1/V 
AgN0 3 . To each flask add 12 drops HN0 3 (1 + 1) and 30 ml H 2 0. 
To one flask add slowly, with const shaking, 30 mL KCN soln 
(3.1 g in 1 L H 2 0). Dil. to vol., shake well, and filter thru dry filter. 
To 100 mL filtrate add 3 mL HN0 3 and 5 mL ferric indicator, 
6.019(e), and titr. with 0.1/V KSCN. 

Pipet 25 mL HCHO distillate into small beaker contg 30 mL of 
the KCN soln, mix well, and add slowly, with const shaking, to 
second flask contg the acidified AgN0 3 soln. Dil. to vol. with 
H 2 0, filter, acidify 100 mL filtrate with 3 mL HN0 3 , and titr. with 
the KSCN soln, using FeNH 4 (S0 4 ) 2 indicator. 

Difference between mL KSCN soln used in these 2 titrns x 2 
= mL 0.1/V KSCN equiv. to HCHO. Calc. % HCHO present. 1 mL 
0.1/V KSCN - 3.003 mg HCHO. 



Ethion (0,0,0',0-Tetraethyl S,S -methylene bis- 
phosphorodithioate) 

High Performance Liquid Chromatographic Method (92) 
Official First Action 

(Applicable to dry and liquid formulations contg ethion as only 
active ingredient.) 

6.352 Apparatus 

(a) Liquid chromatograph. — Waters Associates with Model 
6000A pump, or equiv., with 254 nm UV detector (Waters 
Associates, Inc). Typical operating conditions: eluant flow rate 
1 mL/min (ca 1100 psi), chart speed 0.25 in./min, detector 
sensitivity 0.2 A unit full scale, ambient temp, injection vol. 10 
fxL. Adjust operating conditions to elute ethion peak in 6±2 min. 
Column condition and H 2 content of MeOH eluant can change 
retention times. Ethion peak must be sepd completely from 
internal std peak which normally elutes in ca 7 min (Waters C 18 
column). 

(b) Liquid chromatographic column. — Either (7) Waters 
/xBondapakC 18 , 300 x 3.9 mm id; or (2) DuPont ODS Permaphase, 
0.5 m x 2.1 mm id. 



6.353 Reagents 

(a) Eluant— Either (1) degassed MeOH~H z O (90+10), UV cutoff 
<230 nm, or (2) degassed acetonitrile~H 2 (40+60), UV cutoff 
<230 nm. 

(b) Light mineral oil. — USP, viscosity 38.1 centistokes at 37,8°. 

(c) Internal std soln. — (/) For Waters column. — Accurately 
weigh ca 0.24 g pentachloronitrobenzene (PCNB), ref. grade, 
with no interfering peaks on HPLC, into 200 mL vol. flask. Dil. 
to vol. with MeOH and mix. (2) For DuPont column. — Using 
CH 3 CN as solv., vary amt PCNB in internal std to give peak ht 
approx. same as ethion peak. 

(d) Ethion std soins.-~{1) For Waters column.— Stock soln. — 
Accurately weigh amt of std equiv. to 250 mg ethion, 95+% 
pure (available from Chemical and Biological Investigations, 
Environmental Protection Agency, Beltsville, MD 20705) into 25 
mL vol. flask, dil. to vol. with MeOH, and mix. Working soln.— 
Pipet 10 mL stock soln into 50 mL vol flask, pipet 10 mL internal 
std soln, (c)(7), into flask, dil. to vol. with MeOH, and mix. Prep, 
std and samples daily. (2) For DuPont column. — Prep, as above, 
using CH 3 CN instead of MeOH. (3) For oil formulations. — Pipet 
10 mL 1% stock soln (7) or (2) into 50 mL vol. flask contg ca 
same wt of light mineral oil as sample. Add 20 mL MeOH (or 
CH 3 CN) and proceed as in 6.354(c) beginning with "Stopper and 
agitate . . ." 



6.354 



Preparation of Sample 



(a) Dry powder. — Accurately weigh sample contg ca 100 mg 
ethion into 250 mL g-s flask. Pipet in 40 mL MeOH (or CH 3 CN) 
and 10 mL internal std soln. Shake 30 min on mech. shaker and 
centrf. to sep. phases. 

(b) Liquid concentrates. — Prep, sample as in 6.353(d). 

(c) Oil formulations. — Accurately weigh sample contg ca 100 
mg ethion into 50 mL vol. flask. Add 30 mL MeOH (or CH 3 CN). 
Stopper and agitate vigorously 1 min, with side to side action, 
keeping mixt. in main body of flask. Pipet in 4 mL H 2 and repeat 
vigorous mixing 1 min. Dil. to approx. vol. with MeOH (or 
CH 3 CN). Cool to ambient temp and dil. to vol. Mix thoroly by 
inverting 10 times and swirling vigorously each time. Centrf. to 
sep. phases. 



AOAC Methods (1980) 



Malathion 



113 



6.355 



Determination 



Use high-pressure liq. syringe or sample injection loop to 
inject 10 /u,L portions of std until 2 peak ht ratios agree within 
±1%. Alternately inject two 10 /xL portions each of sample and 
std solns. Measure peak hts and calc. av. peak ht ratios for both 
std and sample. Adjust attenuation or amt injected for convenient 
size peaks (60-80% full scale). Measure peak hts from baseline 
between ethion and internal std peaks. 

% Ethion = (/?//?') x (W'/W) x P, 

where R and /?' = av. peak ht ratios for sample and std, resp.; 
W = mg ethion in working std soln (ca 100 mg); W - mg 
sample in final diln; and P = % purity of std. 



Formothion [S-[2-{Formyl methylamino)-2-oxoethyl] 

0,0-dimethyl phosphorodithioate; 0,0-Dimethvl 

S-(A/-formyl-2-mercapto-A/-methYlacetamide) 

phosphorodithioate] {93) — Official Final Action 



C/PAC-AOAC Method 



6.356 



Reagents 



(a) So/vent I. — Toluene contg 2% Ac 2 0. 

(b) Solvent II.— Hexane-acetone (2 + 1) plus 2% Ac 2 0. 

(c) Internal std soln. — Prep, soln contg ca 100 mg, accurately 
weighed, of ethion/mL solv. I. Ethion must be >95% pure and 
contain no impurities interfering at formothion retention time. 

(d) Reference std soln. — Accurately weigh ca 500 mg For- 
mothion Ref. Std (Sandoz Ltd, Agrochemical Division, CH4002 
Basel, Switzerland) into 50 mL vol. flask, add 5.0 ml_ internal std 
soln, and dil. to vol. with solv. I. 



6.357 



Apparatus 



(a) Gas chromatograph.—{Var\ar\ Aerograph 1520, or equiv.) 
With flame photometric detector (Tracor, FPD 100AT, or equiv.), 
automatic injector (Hewlett-Packard 7600 A, or equiv.), integrator 
(Infotronics CRS 104, or equiv.), and effluent splitter at column 
end with ratio 1:100-1:1000 in favor of outlet. Use glass spiral 
column, 1.0 m x 3.6 mm id, packed with 3% OV 225 on 80-100 
mesh Chromosorb W-HP. Operating conditions: temps (°) — 
oven 210, injector and detector 220; N carrier gas 60 mL/min; 
no. theoretical plates for ethion is ca 2000. Alternatively, flame 
ionization may be used. Conditions are same, except effluent 
splitter is not necessary. 

(b) Bottles. — 50 mL with Mininert valve, or equiv. inert system 
for closure (Pierce Chemical Co.). 



6.358 



Determination 



Accurately weigh well mixed sample contg ca 500 mg for- 
mothion into bottle, (b). Add 5.0 g internal std soln, (c), and dil. 
to 50 mL with solv. I. Close tightly and shake. Transfer 6 ^L soln 
to vial contg 1 mL solv. II. Seal vial with inert valve system. (For 
automatic injections with Hewlett-Packard sampler, dil. in Al 
foil-sealed vials and use Teflon rubber laminated disks as septa.) 
Keep tightly closed. Inject 1.0 /aL dild mixt. into column, by- 
passing solv. around detector by using splitter to avoid contam- 
ination and deterioration. Det. appropriate time for splitting by 
test chromatogram. Compds may be identified by retention 
times relative to ethion as 1.00 (ca 4.4 min): formothion 0.50, 
dimethoate (by-product) 0.36. 

Inject 1 fxL aliquots of reference std soln, (d), until ht or area 
ratio of formothion to ethion varies <2% for successive injec- 
tions. Precede and follow each sample by reference std soln 



and make 3 sep. detns with all peak area ratios of reference std 
solns within ±2% of first accepted values. 

% Formothion = W x H x f x P/W x H' r 
where W and W - mg sample and internal std, resp.; H and H' 
= peak hts or areas of formothion and internal std, resp.; P = 
% formothion in reference compd; 

f - correction factor = w x h'/w' x h; 
where w and w' = mg formothion ref. std and internal std, resp., 
and h and h f = peak hts or areas of formothion and internal std, 
resp. 

Malathion (O^O-Dimethyl Dithiophosphate 
of Diethyl Mercaptosuccinate) 

[Caution: See 51.011 and 51.061.) 

Argent/metric Method [94) — Official First Action 

6.359 Principle 

Malathion is cleaved in alk. soln to dimethyl phosphorodi- 
thioate ion which forms insol. ppt with Ag ion. Pos. bias may 
be encountered. 

6.360 Reagents 

(a) Potassium hydroxide soln.^N. Dissolve 28 g KOH in 500 
mL alcohol. 

(b) Silver nitrate soln.—OAN. Prep, as in 50.027 and stdze 
against primary NaCI as in 6.363. 

(c) Cellulose powder. — Reeve Angel No. CF-11. 



6.361 



Apparatus 



(a) Potentiometer.— Recording potent iometric titrator, oper- 
ated in derivative mode (Metrohm Models E336 thru E576, or 
equiv.; available from Brinkman Instruments), or pH meter with 
mv scale. 

(b) Glass reference electrode with Ag/AgCI internal ele- 
ment—Corning Glass Works, No. 476022. 

(c) Silver billet electrode. — Beckman Instruments, No. 39261. 

(d) Chromatographic tube. — 30 cm x 13.5 mm id. 

6.362 Preparation of Samples 

(a) Technical grade malathion or malathion emuisifiable con- 
centrates.— -Place 2 g cellulose powder in 50 mL beaker, add 0.6 
g H 2 dropwise, and mix thoroly. Place plug of glass wool in 
bottom of chromatgc tube and pack with the wetted cellulose 
powder. Compress column to 5 cm with glass rod. Wet column 
with 5 mL hexane. 

Accurately weigh sample contg 0.5±0.1 g malathion into 250 
mL beaker, add 50 mL hexane, and stir 10 min, using mag. 
stirrer. Transfer to column and collect eluate in another 250 mL 
beaker. Rinse original beaker with three 20 mL portions hexane 
and pour each thru column. Carefully evap. eluate just to 
dryness. Add 50 mL alcohol or isopropanol and stir mag.; add 
20 mL 1A/ KOH and stir 1 min more. Add 50 mL H 2 and 20 mL 
2/V HN0 3 , and stir. 

(b) Powder formulations. — Accurately weigh sample contg 
0.5±0.1 g malathion into 250 mL beaker, add 50 mL hexane, 
cover with watch glass, and stir mag. 20 min at high speed 
without splashing. Filter thru fiber glass paper in 4.25 cm 
buchner and quant, recover filtrate. Thoroly wash beaker and 
funnel with two 20 mL portions hexane. Quant, transfer filtrate 
to 250 mL beaker and evap. hexane just to dryness. Add 50 mL 
alcohol or isopropanol and stir mag.; add 20 mL 1/V KOH and 
stir 1 min more. Add 50 mL H 2 and 20 mL 2/V HN0 3 , and stir. 



114 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.363 



Determination 



(a) pH meter. — Immerse electrodes in soln and set pH meter 
to read absolute mv. Titr. with 0.1/V AgN0 3 soln until meter 
reads ca 520 mv (within 1-2 mL of end point). Continue titrn by 
dropwise addn of titrant to end point, 425 mv. 

(b) Recording potentiometric titrator. — Set instrument in de- 
rivative mode and titr. at 2.0±0.2 mL 0.1/V AgN0 3 soln/min to 
within ca 2 mL of expected end point. Decrease rate to 0.7 ±0.1 
mL/min and continue titrn. Take first inflection in titrn curve as 
end point. 

% Malathion = (V x N x 33.04)/W, 
where V = mL AgN0 3 soln, N = normality of AgN0 3 soln, and 
W = g sample. 

Colorimetric Method [95) — Official First Action 
(Caution: See 51.018, 51.040, 51.041, 51.043, and 51.051.) 



6.364 Principle 

Malathion, 5-(1,2-dicarbethoxyethyl) 0,0-dimethyl phospho- 
rodithioate, is decomposed by alkali in alcohol to l\la 0,0- 
dimethyl phosphorodithioate (NaDMTA), Na fumarate, and al- 
cohol. NaDMTA is converted to Cu +2 complex sol. in cyclohexane 
with formation of intense yellow compd whose intensity is 
proportional to concn of 0,0-dimethyl phosphorodithioic acid 
and which is measured colorimetrically at 420 nm. Pos. bias 
may be encountered. 



6.365 



Precautions 



Vol. of nonaq. solns is highly temp, dependent. Maintain all 
reagents at uniform temp. 

All glassware must be clean and dry. Rinse tubes and pipets 
with MeOH and oven dry before use. After use, rinse all 
glassware in contact with Cu reagent with acetone before 
washing to prevent contamination in future analyses. If diffi- 
culties are still encountered, use 1% HCI in MeOH as wash prior 
to oven drying. 

DeX.A of blank against H 2 whenever new reagents are prepd. 
Deviation from range 0.010-0.020 A units, using 1 cm cells, 
indicates either contamination of glassware or reagents, or 
reagents of improper concn. 



6.366 



Reagents 



(a) Cyclohexane. — Pass thru column of activated silica gel or 
Al 2 3 , activity grade I. Accuracy of assay depends on stability 
of Cu complex which undergoes oxidn-reduction reaction cat- 
alyzed by strong proton donors. Net result is fading of developed 
color. Polar impurities in cyclohexane contribute to this problem 
and qual ity of solv. must be checked as follows before continuing 
with analysis: Perform detn, 6.368, with std soln (b)(7) or {2) 
and det. A at 2 min and again at 12 min. Calc. fade rate (f/?) as 
follows: 

FR = [IAz min ~A n min ) X 100]/H 2 min X 10) 

Fade rate should be ^0.5%/min for std or samples. If fade rate 
exceeds that limit, pass cyclohexane thru silica gel or Al 2 3 
column contg ca 100 g adsorbent/2 L cyclohexane to be treated. 
Ratio of ht of adsorbent bed to its diam. should be >5 and flow 
should be *s3 mL/min. Retest cyclohexane after treatment to 
det. its acceptability. Vol. of cyclohexane that can be treated in 
this manner will depend on extent of contamination. Check 
purity periodically. 

(b) Analytical stds. — Use either malathion or KDMTA, anal, 
grade of known purity (available from American Cyanamid Co.), 



for std soln. Use of malathion will provide assurance that quant, 
elimination reaction is taking place. Store malathion in refrig- 
erator, warming to room temp, before use. Store KDMTA in 
desiccator, avoiding elevated temp. 

(7) Malathion std solns. — Accurately weigh 130-170 mg mal- 
athion into tared 50 mL vol. flask. Dissolve in and dil. to vol. 
with cyclohexane. Store in refrigerator; use at room temp. (2) 
Potassium 0,0-dimethyl phosphorodithioate {KDMTA) std 
solns. — Accurately weigh 80-120 mg KDMTA into tared 50 mL 
vol. flask. Dissolve in and dil. to vol, with acetone. Mix. Store in 
tightly stoppered flask. 

(c) Copper reagent— Dissolve 410-430 mg cupric naphthen- 
ate (ICN-K&K Laboratories, Inc., No. 8172) or 8% Cu Nap-All liq. 
(Mooney Chemicals Inc., 2301 Scranton Rd, Cleveland, OH 
44113) in 100 mL cyclohexane. 

(d) Ethyl acetate.— Contg ^0.2% H 2 and with acidity 
=£0.005% expressed as HOAc. 

(e) Sodium hydroxide soln, — ^N. Dissolve 4 g carbonate-free 
NaOH in 100 mL absolute alcohol or absolute ethanol denatured 
with 0.5% benzene. 

(f) Acetonitriie. — Bp 80-82°. Pass thru column of silica gel, 
discarding yellow first portions of eluate and collecting colorless 
eluate. pH of 10% aq. soln should be 5-7. 

6.367 Preparation of Sample 

(a) Technical materials and emulsifiable concentrates. — Ac- 
curately weigh sample contg 130-170 mg malathion into tared 
50 mL vol. flask. Dissolve in and dil. to vol. with cyclohexane. 
Stopper flask and mix well. 

(b) W enable powders and dusts. — Accurately weigh sample 
contg 130-170 mg malathion on tared weighing paper. Transfer 
to 8 oz narrow-mouth bottle fitted with Vinyl ite-lined screw caps. 
Add 100 mL cyclohexane or acetone, using vol. flask. Place 
sample 10 min on reciprocating shaker set for moderate agita- 
tion. Let solids settle or centrf/ if necessary. Alternatively, use 
150 mL g-s erlenmeyer and mag. stirrer. 

Cyclohexane will not quant, ext malathion from powders 
formulated with bentonite (montmorillonite, AI 2 (Si 4 O 10 )(OH) 2 ), 
and possibly with other carriers. In these cases, ext malathion 
with CH 3 CN or tetrahydrofuran. If CH 3 CN is used, evap. ext to 
dryness in rotary evaporator under vac. at ^50°. Similarly evap. 
appropriate vol. CH 3 CN for reagent blank. Carefully dissolve 
residue in cyclohexane and proceed with detn. If tetrahydrofuran 
is used, proceed as directed, incorporating tetrahydrofuran 
reagent blank. Use alternative extn technics whenever it is not 
known whether method will give adequate extn. 



6.368 



Determination 



Pipet 5 mL aliquots from (a) or 10 mL aliquots from (b) and 
appropriate dild stds to sep. 50 mL vol. flasks. Prep, reagent 
blanks by transferring 5 or 10 mL aliquots cyclohexane to 50 mL 
vol. flask. Use acetone in reagent blank if KDMTA is std. 

At 1 min intervals, add 2 mL 1/V ale. NaOH to each flask in 
sequence, rinsing any sample adhering to neck or sides of flask 
into liq. in flask. Mix well by swirling. Stopper flasks and let 
stand 10 min. Do not have >9 flasks in a series. After 10 min, 
slowly dil. each to vol. with EtOAc, gently swirling flask during 
addn. {Voluminous ppt will form at this time.) Stopper flask and 
mix well. Complete this process for each flask before adding 
EtOAc to next. Let stand 10-20 min with occasional mixing. 

Remix contents of flask and pour portion into centrf. tube. 
Stopper tube and centrf. 5 min. (Supernate should be clear; 
recentrf. if necessary.) Transfer 5 mL to 50 mL vol. flask, taking 
care not to transfer any pptd NaOAc which may remain on 
surface of soln after centrfg. 



AOAC Methods (1980) 



Malathion 



115 



Add 35-40 mL cyclohexane to reagent blank followed by 2 
mL Cu reagent and immediately dil. to vol. with cyclohexane. 
Stopper flask and mix. Use this soln to zero spectrophtr, using 
1 cm cells at max. A, ca 420 nm. Proceed as above for sample. 
Det. A against reagent blank exactly 2 min after adding Cu 
reagent. Slight haze may appear on initial diln with cyclohexane. 
If it does not clear after adding Cu reagent, check for presence 
of excessive H 2 in analysis. 

Water can be introduced by EtOAc, acetone, CH 3 CN, alcohol, 
tetrahydrofuran, or contaminated glassware. Correct this con- 
dition before proceeding with analysis. 



6.369 



Calculations 



(a) Absorptivity of samples (a). — 

(1) Tech. malathion and emulsifiable cones 

a - (A x 50 x 50 x 50)/(mg sample x 5 x 5) 
= (A x 5000)/mg sample 

(2) Wettable powders and dusts 

a = (A x 100 x 50 x 50)/(mg sample x 10 x 5) 
= (A x 5000)/mg sample 

(b) Absorptivity of stds (a'). — 

(1) Malathion: Use formula in (a)(7). 

(2) Potassium 0,0-dimethyl phosphorodithioate 

a' - [{A x 50 x 50 x 50)/(mg sample x 5 x 5)] x 0.5939 
= (A x 2969)/mg sample, 
where 0.5939 is factor to convert K salt to equiv. wt malathion. 
(In general, a of stds are in range 17.2-17.6, corrected for quality 
of std, at concn 1 mg/mL in 1 cm cell.) 

(3) % Malathion = {a x % purity of std)/a' 

6.370 Preparation of Standard Curve [Optional) 

Transfer 3, 4, 5, and 6 mL aliquots centrfgd EtOAc soln of std, 
6.368, par. 3, into sep. 50 mL vol. flasks. Proceed with color 
development, and plot A against wt malathion in aliquot. Use 
std curve with considerable caution, since fade rate may vary 
from day to day, depending on solvs used for analysis. Std 
curve is not appropriate where fluctuations in temp, occur in 
laboratory environment. 



6.371 



Interferences 



Test for free 0,0-dimethyl phosphorodithioic acid or other 
interfering components in sample ext as follows: Transfer 2 
drops ext to test tube, dil. with ca 10 mL cyclohexane, add 8 
drops Cu reagent, and mix. Development of discernible yellow 
in soln indicates necessity of obtaining sample-blank correction. 

Obtain sample-blank correction as follows: Transfer 5 or 10 
mL aliquot sample to 50 mL vol. flask and dil. to vol. with EtOAc. 
(Do not add ale. NaOH soln.) For color development, transfer 5 
mL dild sample soln and continue as in 6.368. Correct A for 
base-treated sample and proceed with calcn. 



Gas Chromatographic Method (96) — Official First Action 

6.372 Apparatus 

(a) Gas chromatograph. — With glass column, on-column in- 
jection system, flame ionization detector, and electrometer with 
sensitivity of s=10 -11 amp driving 1 mv recorder. Drift should be 
<1%/hr. Totally solid state amplifier with FET input is recom- 
mended. Electronic digital integrator or computer calcd area 
measurements must be used. Integrator should have independ- 
ent controls for selection of up and down slope sensitivities so 
that start and stop integration points can be selected. Automated 
sample injection system contributes significantly to precision. 
Hewlett-Packard Model 7600 is suitable when equipped as 



described. Equiv. instrumentation may be used but may require 
modification of operating conditions to obtain good peak shape, 
adequate resolution, and appropriate retention times. 

Typical conditions for Hewlett-Packard Model 7600 (instru- 
ment may have to be adjusted to give complete resolution of 
well shaped peaks): Cycle timers (min): analysis and stop 
integrate, 16; range, 10 3 ; temps (°): oven 180, injection port 200, 
flame detector 300; gas flow rotameters (mL/min): H 35, air 
425, He carrier gas 30; integrator settings (adjusted so that 
deflections on slope meter do not exceed ±50% before injec- 
tion): noise suppression max., slope sensitivity up and down 
0.1, BL reset delay 0.15, area threshold 1000; retention times 
(min): malathion 10, internal std 6, min. time between malathion 
and internal std 3.5. 

(b) Column. — Borosilicate glass tube 1.22 m (6') x 4 mm id, 
6 mm od, bent to fit chromatograph and packed with 5% SP- 
2401orOV~210onGas-ChromQorSupelcoport(100/120mesh). 
Can be purchased as prepd packing from Supelco, Inc., (specify 
"Pesticide Grade"); Alltech Associates, 202 Campus Dr, Arling- 
ton Heights, IL 60006; and Applied Science Laboratories, Inc. 
Use exclusively for malathion analysis. 

(c) Glass wool. — Silane treated. (No. 14502, Applied Science 
Laboratories, Inc.) 

(d) Syringes.— 10 fiL, Series 700, Hamilton Co. 

(e) HI-EFF Fluidizer. — Applied Science Laboratories, Inc. 



6.373 



Reagents 



(a) Internal std soln. — 1.2% m-Diphenoxybenzene in CHCI 3 . 
Must not contain any impurities which elute at or near malathion 
peak. Bring soln to consistent temp, above ambient (e.g. 25°) 
before taking aliquots. 

(b) Malathion std solns. — Accurately weigh ca 170, 200, and 
230 mg malathion std (anal, grade, available from American 
Cyanamid Co.) into sep. preweighed 25 mL vol. flasks. Add by 
pipet 5 mL internal std soln and dil. to vol. with CHCI 3 . Label 
A, B, and C. Soln B is working std soln for detn; solns A 
and C are used for linearity check and to guard against weighing 
error in prepn of working std soln. Solns are stable ca 4 weeks 
if kept tightly sealed in refrigerator. Warm to room temp, before 
use. Soln B can be prepd independently of solns A and C, if 
conditions of linearity check are met. 



6.374 Preparation and Conditioning of Column 

Weigh 6.25 g of trifluoropropylsilicone (SP-2401 orOV-210) 
in 250 mL beaker and dissolve in 125 mL EtOAc. Stir to obtain 
vortex and add 25 g solid support (Gas-Chrom Q or Supelcoport, 
100/120 mesh) with continued agitation. Filter slurry thru What- 
man No. 1 paper, or equiv., on buchner, using gentle vac. to 
minimize evapn of solv. Continue filtration until drop rate is ca 
1/sec. Transfer packing to HI-EFF Fluidizer, connect source of N 
thru pressure reducer to base, and place fluidizer on controlled 
temp, hot plate set for 75°. Continue gas flow until solv. vapors 
can no longer be detected by odor, taking care that packing is 
not blown out top of fluidizer. 

To pack column, attach 75 mm funnel to exit end of prebent 
glass tube. Tap tube with pencil or small wooden rod, and add 
prepd packing in small amts until exit end is filled to ca 15 mm 
from end. Move funnel to entrance end of column. Insert pledget 
of silane-treated glass wool in exit end and attach source of 
moderate vac. to this end. Continue to add packing slowly with 
tapping until tube is filled to ca 20 mm from entrance end. Insert 
pledget of silane-treated glass wool in entrance end, compress- 
ing it only enough to hold it in place. 

Condition column with He carrier gas flowing at 30 mL/min 



116 



6. Pesticide Formulations 



AOAC Methods (1980) 



3=15 hr (overnight) at 255° or ca 20° below max. temp, recom- 
mended for iiq. phase. Exit end of column should not be 
connected to detector during this conditioning. 

Connect exit end of column to detector, adjust controls to 
conditions given in 6.372(a), and let instrument come to equi- 
librium. Inject 3 fit aliquots std soln C until ^3 consecutive 
injections give response ratios agreeing within 2%. 

6.375 Linearity Check 

Check gas chromatograph for linearity at least weekly, when- 
ever new std solns are prepd, and whenever column, new or 
used, is newly installed in instrument. 

Using digital integration for peak area measurements, det. 
appropriate attenuation setting and injection aliquot (2-4 (xL) of 
std soln B to give area count of ^100,000 counts (optimum 
electrometer output with acceptable noise level). Use conditions 
so detd for all samples and stds in series. 

Inject triplicate aliquots of detd vol. of std solns A, B, and C 
into chromatograph, det. response ratio for each, and aver- 
age ratios for each soln. Divide av. ratio for each soln by 
corresponding malathion content in mg. Ratio/mg should agree 
within 2%. Failure to meet this specification indicates either 
weighing error in prepn of a std soln or instrumental difficulties 
which must be corrected before proceeding with analysis of 
samples. 

6.376 Preparation of Sample 

(Analyze samples at least in duplicate) 

(a) Liquid formulations and technical materials. — Accurately 
weigh sample contg ca 200 mg malathion into preweighed 25 
ml_ vol. flask. Pipet in 5 ml_ internal std soln, dil. to vol. with 
CHCI 3 , and mix well. 

(b) Solid formulations containing 10% or more of malathion. — 
Accurately weigh sample contg ca 1.0 g malathion and transfer 
to 200-250 ml_ (8 oz) bottle. Pipet in 50 mL CHCI 3 , stopper tightly, 
and shake on reciprocating shaker 30 min. Let settle ca 15 min; 
if not clear, centrf. Layer of solids will float at interface. Avoid 
entrainment of particles by exerting pos. pressure from bulb on 
pipet while it is carefully inserted into soln for removal of aliquot. 
Particles in final soln can clog syringe needle. Transfer 10 mL 
aliquot clear soln to 25 mL vol. flask, pipet in 5 mL internal std 
soln, dil. to vol. with CHCI 3/ and mix well. 

(c) Solid formulations containing less than 10% malathion. — 
Accurately weigh sample contg ca 400 mg malathion and 
transfer to 500 mL (16 oz) bottle. Add exactly 200 mL CHCI 3 and 
shake 30 min on reciprocal shaker. Let settle, observing precau- 
tions given in (b). Pipet 100 mL aliquot to 500 mL r-b flask and 
evap. to dryness. Pipet in 5 mL internal std soln and 20 mL 
CHCU, swirl to dissolve residue, and mix well. 



6.377 



Determination 



Inject duplicate aliquots of appropriate vol. of std soln B as 
detd in linearity check, 6.375. Response ratios should agree 
within 2%; if not, repeat with 2 more injections. Failure to meet 
specification with second pair of injections indicates instru- 
mental difficulties which must be resolved before proceeding 
with analysis. 

Inject duplicate aliquots of each sample soln of same vol. as 
std soln. Average response ratios for each sample. Precision 
considerations stated for std soln also apply to sample soln 
injection response. 

Inject duplicate aliquots std soln B after every 2 sample solns. 
Average response ratios of stds immediately before and after 
sample solns. Use this av. to calc. malathion content of the 2 
sample solns. 



Each detn of av. response ratio for std soln B should yield 
value within 2% of previously detd value. Failure to meet this 
specification indicates instrumental drift which must either be 
corrected or compensated for by more frequent measurements 
of response of std soln B. In extreme cases, follow each sample 
injection with std injection but this would indicate an instability 
which should be corrected at once. 

6.378 Calculations 

For each sample injection, calc. response ratio: 

R - area of malathion peak/area of internal std peak 
% Malathion = (R/R'J x (W'/W) x P x D 
where R' and R = av. response ratio for std soln B and sample 
soln, resp; W and W = g malathion std and sample, resp.; P 
~ % purity of malathion std; and D = diln factor (1 for liqs; 
(50/10X25/25) = 5 for solids ^10% malathion; and 
(200/100K25/25) = 2 for solids <10% malathion). 



Pa rath ion 
Gas Chromatographic Method [97) — Official First Action 

(Not applicable to dusts and powders) 



6.379 



Standard Solutions 



(a) Dipentyl phthalate internal std soln. — Dissolve 2.0±0.1 g 
dipentyl phthalate (Eastman Kodak Co., No. P2473, or equiv.) in 
CS 2 and dil. to 500 mL with CS 2 , 

(b) Parathion std soln. — Accurately weigh ca 125mgparathion 
(Monsanto Chemical Co., or equiv.) into 50 mL g-s erlenmeyer, 
pipet in 25 mL internal std soln, and mix thoroly. 

6.380 Preparation of Sample 

Accurately weigh sample contg ca 125 mg parathion into 50 
mL g-s erlenmeyer. Pipet in 25 mL internal std soln and mix 
thoroly. 



6.381 



Gas Chromatograph 



See 6.402. Column should have ^1200 theoretical plates for 
parathion. Vary attenuation and injection vol. (1-2 (jlL) so that 
peak hts of parathion and dipentyl phthalate are 60-80% full 
scale on 1 mv recorder. Retention times for parathion and 
dipentyl phthalate are 6-8 and 8-10.5 min, resp. 



6.382 



Determination 



Proceed as in 6.403, except substitute parathion for Me 
parathion and dipentyl phthalate for p,p '-DDE. 



6.383 



Calculations 



Proceed as in 6.404, except substitute parathion for Me 
parathion and delete/ 7 from equation. 



High Pressure Liquid Chromatographic Method [97) 
Official First Action 

(Not applicable to dusts and powders) 

6.384 Apparatus 

(a) Liquid chromatograph. — See 6.405(a), except use el u ant 
flow rate of 1.5 mL/min (ca 800 psi). 

(b) Liquid chromatographic column. — See 6.294(b). 



AOAC Methods (1980) 



Parathion 



117 



6.385 



Reagents 



6.390 



Preparation of Standard Curve of p-Nitrophenol 



(a) Choroform.—See 6.406(a). 

(b) E/uant — Stir 500 ml_ CHCI 3 on mag. stirrer 3-4 min under 
moderate vac. (ca 350 mm Hg). 

(c) Internal std soln, — Accurately weigh ca 110 mg benzo- 
phenone (MC/B Manufacturing Chemists, No. BX0410, or equiv.) 
into 250 ml_ vol. flask, and dissolve and dil. to vol. with CHCI 3 . 

(d) Parathion std solns. — (7) Stock soln. — 1500 ju,g/mL Ac- 
curately weigh ca 75 mg anal, grade parathion (Monsanto 
Chemical Co., or equiv.) into 50 mL vol. flask, and dissolve and 
dil. to vol. with CHCI 3 . (2) Working soln.— (150 /xg parathion + 
44 /xg benzophenone)/mL Pipet 5 mL stock soln and 5 mL 
internal std soln into 50 mLvol. flask, and dil. to vol. with CHCI 3 . 



6.386 



Preparation of Sample 



Accurately weigh sample contg ca 75 mg parathion into 50 
mL vol. flask, and dissolve and dil. to vol. with CHCI 3 . Pipet 5 mL 
sample soln and 5 mL internal std soln into 50 mL vol. flask, and 
dil. to vol. with CHCI 3 . 

6.387 Determination 

Proceed as in 6.408, except substitute parathion for Me 
parathion and benzophenone for acetophenone, and delete F 
from equation. Retention times for parathion and benzophenone 
are 4.0-5.5 and 7-9 min, resp. 



Volumetric Method [98) — Official First Action 

(Applicable to dusts and powders only. Caution: See 51.041.) 

6.388 Apparatus 

(a) Photoelectric colorimeter. — With filter to give max. T be- 
tween 400 and 450 nm. Spectrophtr set at 405 nm may also be 
used. 

(b) Potentiometer. — With adapter for outside Pt and calomel 
electrodes. Dead-stop end point equipment may also be used. 



6.389 



Reagents 



(a) Zinc dust. — Low in Fe. 

(b) Suifanilic acid. — Anhyd. recrystd material. Check purity by 
N detn. 

(c) p-Nitrophenol.— W\p 112-113°. 

(d) Sodium nitrite std soln. — 0.1/V. Stdze weekly. Accurately 
weigh 0.4—0.45 g of the suifanilic acid into 400 mL tall beaker. 
Add 80 mL H 2 0, 10 mL HCI, 30 mL HOAc, and 5 g NaBr. Place 
electrodes and mech. stirrer in reaction mixt. and titr. with the 
0.1/V NaN0 2 . Add in 5 mL portions until within 1 mL of calcd 
end point; then add NaN0 2 soln in 0.1 mL portions until max. 
rise in potential is obtained. At first, 3-5 min is required for 
potential to become const; as end point approaches, espe- 
cially after 0.1 mL addns, reaction should be complete within 1 
min. As alternative, dead-stop end point technic may be used 
(98), or following spot test, adding NaN0 2 soln in 4 drop portions 
near end point: Dip glass rod into soln being titrd and touch rod 
quickly to piece of Kl-starch paper, (e>. End point is reached 
when intense blue-black color appears immediately and can be 
obtained repeatedly during 1 min period without further addn 
of NaN0 2 . 

Normality NaN0 2 soln = g suifanilic acid x 1000/(mL NaN0 2 
x 173.2). 

(e) Starch iodide paper. — Triturate 10 parts starch with 200 
parts H 2 0, bring to bp, and add 1 part Kl. Impregnate strips of 
filter paper with this soln, dry, and preserve in g-s bottles. 



Accurately weigh 100 mg p-nitrophenol, transfer to 1 L vol. 
flask, and dil. to vol. with 0.1/V NaOH. Transfer 2, 4, 6, 8, 10, and 
20 mL aliquots of this soln to 100 mL vol. flasks and dil. each 
soln to vol. with 0.1/V NaOH. Read A of each soln in photoelec. 
colorimeter (400-450 nm) or spectrophtr (405 nm) against H 2 
as ref. Plot A against concn in mg/mL. 

6.391 Preparation of Sample 

Dust preparations and wettable powders. — Transfer weighed 
sample to thimble and ext with 150 mL ether in Soxhlet app. 1 
hr. Transfer ether ext to 250 mL separator, and sep. p-nitrophenol 
and parathion as in 6.392. Det sample size by parathion concn 
as follows: 10%, 6.75 g; 15% 4-5 g; 25%, 2.5-3.5 g. 

6.392 Separation of Parathion and p-Nitrophenol 

Ext ether soln with four (or until ext is colorless) 20 mL 
portions chilled 1% Na 2 C0 3 soin f collecting combined aq. layers 
in 200 mL vol. flask. Transfer ether layer to 400 mL tall beaker, 
rinsing separator with small portions ether. 

6.393 Determination of p-Nitrophenol 

Add 20 mL 1/V NaOH to combined aq. exts and dil. to vol. with 
H 2 0. Measure A of soln as in 6.390 and read concnp-nitrophenol 
in mg/mL from std curve. 
% p-Nitrophenol = (mg/mL) x 200 x 100/(1000 x g sample). 



6.394 



Determination of Parathion 



{Caution: See 51.011, 51.039, and 51.054.) 

Add 35 mL HOAc-HCI mixt. (9+1) to ether soln, 6.392. Add 2 
g Zn dust, cover beaker with watch glass, and gently heat soln 
on steam bath 45 min, or until most of ether evaps and soln is 
colorless. Add 30 mL HCI and heat 10 min longer to complete 
soln of Zn dust. Wash down beaker and watch glass with H 2 0. 
Filter reduced mixt. thru paper and rinse beaker thoroly with 
H 2 0. Dil. to 125 mL and cool to room temp. Add 5 g NaBr (or 
KBr) and titr. with 0.1/V NaN0 2 as in 6.389(d). 

% Parathion = mL NaN0 2 x normality x 29.13/g sample. 

Colorimetric Method {99)—Officiaf First Action 

(Applicable to dusts and powders only) 

6.395 Principle 

Parathion is extd with alcohol and hydrolyzed with KOH to 
form Kp-nitrophenate, which is detd colorimetrically. 



6.396 



Preparation of Standard Curve 



Weigh 60 mg reagent grade p-nitrophenol into 100 mL vol. 
flask, dissolve in alcohol, and dil. to vol. with alcohol. Pipet 10 
mL into 100 mL vol. flask and dil. to vol. with alcohol. Prep, p- 
nitrophenol stds contg 0.3, 0.18, and 0.06 mg/100 mL by 
pipetting 5, 3, and 1 mL aliquots, resp,, of second diln into sep. 
100 mL vol. flasks, adding from pipet 5 mL IN KOH in 50% 
alcohol, and dilg to vol. with 50% alcohol. Measure >4 at 405 nm 
in 1 cm Corex cells against 50% alcohol and plot A against 
concn. 

6.397 Preparation of Sample 

Weigh sample contg ca 10 mg parathion into 250 mL g-s flask. 
Pipet in 100 mL alcohol and shake occasionally during 10 min. 
Filter ca 25 mL into g-s container. 



118 



6. Pesticide Formulations 



AOAC Methods (1980) 



6.398 



Determination of Free p-Nitrophenol 



Pipet 10 imL aliquot of above soln into 100 mL vol. flask and 
dil. to vol. with 50% alcohol. Add 5 drops 1/V KOH in 50% 
alcohol, and measured at 405 nm within 2 min against 50% 
alcohol. Calc. freep-nitrophenol. 



6.399 



Determination of Parathion 



Pipet 5 mL filtered soln into 125 mL g-s flask, pipet in 5 mL 
1/V KOH in 50% alcohol, and add glass beads to prevent 
bumping. Reflux 5=30 min. Cool, and transfer to 100 mL vol. 
flask with 50% alcohol. Dil. to vol. with 50% alcohol and measure 
A as in 6.396. Calc. parathion, using std curve, dilns, and factor: 
Parathion = p-nitrophenol/0.478. Correct for freep-nitrophenol. 

Methyl Parathion 
Gas Chromatographic Method (100) — Official First Action 

6.400 Standard Solutions 

(a) p,p'-DDE internal std soln.— Dissolve 5.0±0.1 g 2,2-bis(p- 
chlorophenyl)-1,1-dichloroethylene(p,p'-DDE, No. 12.389-7, Aid- 
rich Chemical Co., Inc., or equiv.) in CS 2 and dil. to 1 L with CS 2 . 

(b) Methyl parathion std soln. — Accurately weigh ca 125 mg 
Me parathion (Monsanto Chemical Co., 800 N Lindbergh Blvd, 
St. Louis, MO 63166, or equiv.) into 50 mL g-s erlenmeyer, pipet 
in 25 mL internal std soln, and mix thoroly. 

6.401 Preparation of Sample 

(a) Liquid, — Accurately weigh into 50 mL g-s erlenmeyer 
sample contg ca 125 mg Me parathion. Pipet in 25 mL internal 
std soln and mix thoroly. 

(b) Wettable powder.— Accurately weigh into 100-150 mL (4 
oz) round bottle sample contg ca 625 mg Me parathion. Pipet 
in 50 mL CHCI 3 -acetone (9+1 ), cap, and shake mech. 30 min. Let 
settle and pipet 10 mL supernate into 50 mL g-s erlenmeyer. 
Place erlenmeyer in 55° H 2 bath and evap. solv. under stream 
of dry air or N. Pipet in 25 mL internal std soln and mix thoroly. 



6.402 



Gas Chromatograph 



Use instrument equipped with flame ionization detector and 
1.2 m x 4 (id) mm glass column packed with 1.5% SE-30 plus 
1.5% OV-210 on 80-100 mesh Gas-Chrom Q. 

Prep, column by accurately weighing ca 0.12 g SE-30 and ca 
0.12 g OV-210 into 250 mL beaker. Add 50 mL CHCI 3 -acetone 
(3+2), cover with watch glass, and heat on steam bath until 
stationary phases are dissolved. Speed dissoln of SE-30 by 
spreading material on walls of beaker with small spatula or 
stirring rod. Add enough 80-100 mesh Gas-Chrom Q to yield 
1.5% of each phase on solid support. Heat on steam bath, 
stirring frequently until all solv. is removed. Air dry 2-3 hr. Pack 
in column and condition 24 hr at 245° with N or He at 30 mL/min. 
Column should have ^1200 theoretical plates forp,p'-DDE. 

Typical operating conditions: temps (°)— inlet 210, column 
180±10, detector 250; N or He carrier gas, 55-75 mL/min; air 
and H as specified by manufacturer; attenuation and injection 
vol. (1-2 ^L) varied so that peak hts of Me parathion and p,p'- 
DDE are 60-80% full scale on 1 mv recorder. Retention times 
for Me parathion and p,p'-DDE are 3.5-5.5 and 6-8 min, resp. 



6.403 



Determination 



Inject aliquots of std soln until peak ht ratio of Me parathion : 
p,p'-DDE varies ^1% for successive injections. Then make 
duplicate injections of sample followed by duplicate injections 
of std. Peak ht ratios of stds must be within ±1% of first accepted 



std values or repeat series of injections. Repeat for addnl 
samples. 

6.404 Calculations 

Calc. peak ht ratios for both duplicate std injections preceding 
and following samples. Average the 4 values (/?'). Calc. and 
average peak ht ratios of the 2 samples (/?). 

% Me parathion = (/?//?') x [W'/W) x F x P, 
where W and W = mg sample and std, resp.,/ 7 = 1 for liq. and 
5 for wettable powder samples; and P = % purity of std. 



High Pressure Liquid Chromatographic Method [701) 
Official First Action 



6.405 



Apparatus 



(a) Liquid chromatograph.— Waters Model ALC 202/GPC 204 
(Waters Associates, Inc.), or equiv., with 254 nm UV detector 
and 10 mv recorder. Typical operating conditions: eiuant flow 
rate, 1.2 mL/min (ca 700 psi); detector sensitivity, 0.16/4 unit 
full scale; temp., ambient; valve injection vol., 10 /liL. 

(b) Liquid chromatographic column. — See 6.294(b). 

(c) Chromatographic tubes. — Glass, 900 x 25 (id) mm, with 
coarse porosity frit in bottom (SGA Scientific, Inc., No. JC-2650, 
or equiv.). 

6.406 Reagents 

(a) Chloroform.— Alcohol-free with <0.01% H 2 (Burdick & 
Jackson Laboratories, Inc., distd in glass, or equiv.). 

.(b) Silicic acid-water.— 75% (w/v). Add 25 mL H 2 to 75 g 
silicic acid (Mallinckrodt Chemical Works, Code 2847, or equiv.), 
and shake until lumps disappear. 

(c) Water-saturated chloroform.— Shake 700 mL CHCI 3 with 
150 mL H 2 2-3 min, and pass thru 900 x 25 mm glass tube 
packed with 100 g silicic acid-H 2 0. 

(d) Eiuant— Blend 200 mL H 2 0-satd CHCI 3 with 300 mL CHCI 3 
on mag. stirrer 2-3 min under moderate vac. (ca 350 mm Hg). 

(e) Internal std soln. — Accurately weigh ca 115 mg aceto- 
phenone (MC/B Manufacturing Chemists, No. AX0164, or equiv.) 
into 250 mL vol. flask, and dissolve and dil. to vol. with CHCI 3 . 

(f) Methyl parathion std solns. — ( /) Stock soln. — 700 ^g/ mL. 
Accurately weigh ca 70 mg anal, grade Me parathion (Monsanto 
Chemical Co., or equiv.) into 100 mL vol. flask, and dissolve and 
dil. to vol. with CHCI 3 . (2) Working soln.— (70 fig Me parathion 
+ 46 fxg acetophenone)/mL. Pipet 5 mL stock soln and 5 mL 
internal std soln into 50 mL vol. flask, and dil. to vol. with CHCI 3 . 

6.407 Preparation of Sample 

Accurately weigh ca 95 mg tech. Me parathion into 100 mL 
vol. flask, or accurately weigh emulsifiable sample contg ca 35 
mg Me parathion into 50 mL vol. flask, and dil. to vol. with 
CHCI 3 . Pipet 5 mL sample soln and 5 mL internal std soln into 
50 mL vol. flask, and dil. to vol. with CHCI 3 . 



6.408 



Determination 



Pump sufficient eiuant thru column to equilibrate system. 
Inject 10 ixL working std soln onto column thru sampling valve, 
and adjust operating conditions to give peak hts 60-80% full 
scale and retention times of 3.5-5.0 and 5.5-8.0 min for Me 
parathion and acetophenone, resp. Repeat injections until ratio 
of Me parathion to acetophenone peak hts is within ±1% of 
previous injection. Without changing conditions, alternately 
inject 10 /xL aliquots of working std soln and duplicate 10 (jlL 
aliquots of sample soln until peak ht ratios for sample soln vary 



AOAC Methods (1980) 



Phorate 



119 



^1% for successive injections. Average last 2 peak ht ratios for 
sample and for std, resp., and calc. % Me parathion. 
% Me parathion - (/?//?') x (W'/W) x (P/F), 
where R and /?' = av. peak ht ratios of Me parathion to 
acetophenone for sample and std, resp.; W and W = mg sample 
and std, resp.; P = % purity of std; and F = 1 for tech. and 2 for 
emulsifiable samples. 

Methyl Parathion in Water-based 
Microencapsulated Formulations 

Gas Chromatographic Method {102) — Official First Action 

6.409 Principle 

Me parathion is released from microcapsules by grinding, and 
is extd into CH 3 CN. Dimethoate is added as internal std and 
concn of Me parathion is detd by flame ionization GLC. 

6.410 Apparatus 

(a) Gas chromatograph. — Perkin-Elmer Model 900, or equiv., 
with flame ionization detector, glass lined injection port, 1 mv 
strip chart recorder, and 1.8 m x 2 (id) mm glass column packed 
with 3% OV-17 on 80-100 mesh Supelcoport (Supelco, Inc.). 
Typical operating conditions: temps (°): column 200, injection 
port 225, detector 250; flow rates (mL/min): He carrier gas 35, 
air 400, H optimize for max. sensitivity; sample: 1 fxL CH 3 CIM 
contg dimethoate and Me parathion with retention times of ca 
3 min and 4 min, resp. Injection vol. may be varied to give peak 
hts 50-90% of full scale. 

(b) Sample grinder. — Spex Industries Mixer/Mill No. 8000 
(Spex Industries, Inc., PO Box 798 r Metuchen, NJ 08840) or 40 
ml_ Corning 7726 glass tissue grinder (No. 441969, Corning 
Glass Works). 

(c) Weighing dishes. — With natural Al surface to which sample 
does not stick (Fisher Scientific Co., No. 8-732, or equiv.). 

6.411 Reagents 

(a) Dimethoate. — Cygon® insecticide, anal, grade (obtainable 
from American Cyanamid Co.). 

(b) Methyl parathion. — Anal, grade (obtainable from Mon- 
santo Chemical Co.). 



6.412 



Determination of Correction Factor 



Prep. 2 duplicate std solns by accurately weighing ca 0.1 g Me 
parathion and ca 0.1 g dimethoate directly into same 50 mL vol. 
flask, and dilg to vol. with CH 3 CN. Shake thoroly to dissolve. 
Inject ca 1 ^L each soln into gas chromatograph. Repeat 
injections until ratio of peak hts is reproducible; then record 
peak hts and attenuations for dimethoate and Me parathion. 

CF = (P 6 x W mp )/(P mp x W d ), 
where CF = correction factor, P d = peak ht x attenuation for 
dimethoate, tV mp = g Me parathion, P mp = peak ht x attenuation 
for Me parathion, and W d = g dimethoate. 

Average results for the 2 solns. 

6.413 Preparation of Sample 

Prep, duplicate samples as follows: Thoroly shake sample 
container to assure that slurry of microcapsules is homogene- 
ous. Withdraw ca 1 g sample using medicine dropper while 
stirring. Immediately discharge contents into tared Al weighing 
dish, and record exact wt. Transfer to Mixer/Mill or glass tissue 
grinder, using small amt of CH 3 CN. (If anal, balance can accom- 
modate grinder, sample may be weighed directly in it.) Add ca 
30 mL CH 3 CN and grind ca 4 min. Quant, transfer ground sample 



to 100 mL vol. flask, using CH 3 CN. Accurately weigh ca 0.2 g 
dimethoate in tared Al weighing dish, transfer quant, to vol. 
flask, and dil. to vol. with CH 3 CN. 

6.414 Determination 

Inject ca 1 fxL soln contg sample and internal std into gas 
chromatograph. Record peak hts and attenuations for dime- 
thoate and Me parathion. 

wt % Me parathion - {P mp x W d x CF x 100)/(P d x W), 
where P mp - peak ht x attenuation for Me parathion, W d = g 
dimethoate, CF = correction factor, P d - peak ht x attenuation 
for dimethoate, and W = g sample. 

Analyze duplicate samples and av. results. 



Phorate (Thimet®) (0,0-Diethyl S-(Ethylthio)methyl 
Phosphorodithioate) {103) — Official Final Action 

(Applicable to analysis of 5 and 10% granules. Presence of other 
pesticides and extractable org. materials such as dispersing 
agents, emulsifiers, and solvs requires testing for interference.) 

6.415 Apparatus 

(a) Infrared spectrophotometer. — Capable of measurement in 
7.9-8.6 /Am range; with 0.5 mm cell. 

(b) Chromatographic tube. — 15 x 450 mm with stopcock or 
Ultramax valve (Fischer & Porter Co., Lab Crest Scientific Div., 
Cat. No. 274-019 or 274-100). 

6.416 Reagents 

(a) Phorate reference std. — Purified (obtainable from Ameri- 
can Cyanamid Co.). 

(b) Phorate std soln. — Accurately weigh by difference from 
Smith or Lunge pipet 1.0-1.1 g Phorate Ref. Std into 250 mL 
beaker contg 45 mL CH 3 CN. 

(c) Cyclohexane. — Practical grade. 

(d) Acetonitrile. — Practical grade, bp 82-84°. 

6.417 Preparation of Sample Solution 

[Caution: See 51.011, 51.040, and 51.043.) 

Accurately weigh 20±0.01 g sample of 5% granular material 
(10±0.01 g for 10%). Place small glass wool plug in bottom of 
chromatgc tube, transfer sample to tube, and gently tap sides 
with spatula or rod to settle contents. Place 250 mL beaker under 
column. Add 50 mL CH 3 CN to column and let percolate thru at 
rate of 40-50 drops/min until flow stops. Place beakers contg 
std (from 6.416(b)) and sample solns in shallow H 2 bath at 
30-35° and evap. under gentle stream of air until odor of CH 3 CN 
is no longer detectable. (Sample solns on evapn will change 
from clear to cloudy and then to residue of 2 layers.) Treat 
residue with four 5 mL portions and one 4 mL portion cyclo- 
hexane, quant, transferring cyclohexane layers to 25 mL vol. 
flask. (Keep cyclohexane-immiscible layer in beaker during each 
extn.) Dil. to vol. with cyclohexane. 



6.418 



Determination 



Using hypodermic syringe, fill 0.5 mm cell with prepd std 
soln, and obtain IR spectrum from 7.9 to 8.6 ^m. (With single 
beam instrument, adjust to give 75% T at 8.2 jum with cell contg 
std soln in position.) Using same instrument settings, treat 
prepd sample solns similarly. 

Draw baseline from inflection points 8.10 to 8.48 ^m. Draw 
perpendicular from radiation line thru absorption peak, and 
measure distance from Oto baseline (V) and from Oto absorption 



120 



6. Pesticide Formulations 



AOAC Methods (1980) 



peak (X) in same units. Calc. A = log (Y/X) for sample (A) and 

std W). 
% Phorate = {A/ A') x (wt std/wt sample) x % purity of std. 

6.419 • Sulfoxide {n-Octyl Sulfoxide of * 

Esosafrole) (104) — Official First Action 

Sulfoxide is sepd from solvs, emulsifiers, pyrethrins, and 
other insecticides by silicic acid column chromatgy with suc- 
cessive eluting solns: CHCI 3 , 2% acetone in CHCI 3 , and 10% 
acetone in CHCI 3 . Sulfoxide is removed in last eluate and is detd 
by UV spectrophotometry. See 6.296-6.302, 11th ed. 

Tetraethylpyrophosphate (TEPP) 
(105)— Official Final Action 



{Caution: See 51.041.) 



6.420 



Reagents 

(a) Indicator. — 0.1% aq. soln Me red or chlorophenol red. 

(b) Amberlite IR-4B{0H) [free base form) resin .—Anal, grade. 
Amberlite IR-45, Dowex 3, or equiv., are satisfactory. 



6.421 



Preparation of Resin Column 



Screen resin to remove particles <30 mesh. Slurry 30 g 
screened resin with H 2 0, and pour into 100 mL buret contg small 
plug of glass wool at bottom. Wash resin column with 150 mL 
3% NaOH soln at flow rate of ca 5 mL/min and then rinse with 
H 2 until effluent is acid to phthln, adjusting stopcock of buret 
so flow rate is ca 25 mL/min. Wash with aq. acetone (1+3) to 
displace H 2 0. Column is now ready for use. 

Notes: Because channeling may result if column runs dry, 
keep liq. level ca 2.5 cm above resin bed at all times. Because 
resin tends to pack in column as it adsorbs acidic material, 
expand resin bed after each detn before adding new sample by 
back-washing with acetone (1+3) as follows: Connect large 
funnel to tip of buret with rubber hose, and add the dil. acetone 
from funnel until liq. level reaches top of buret; let resin settle, 
and then let soln flow from buret until surface is 2.5 cm above 
resin bed. Column is now ready to receive next sample. 

After 8-10 samples have passed thru column, regenerate resin 
by repeating original treatment with 3% NaOH soln, H 2 0, and 
acetone (1+3). Washing with dil. acetone must be continued 
until effluent is colorless. 



6.422 



Determination 



{a) In purified or technical grades of tetraethylpyrophosphate 
not mixed with solvent emulsifying agent, etc.— From 5-10 mL 
weighing buret, weigh by difference, to nearest mg, 2.5 g sample 
(1.0 g if tetraethylpyrophosphate content is >50%) into 50 mL 
acetone (1+3) in 125 mL separator. Mix by swirling, and let soln 
stand 1 5 min at 25±2°. Let soln flow thru resin column by gravity 
at ca 25 mL/min, and catch effluent in 250 mL vol. flask. Wash 
separator and column with three 50 mL portions acetone (1+3), 
collecting washings in same flask. Dil. combined effluent to vol. 
with H 2 0, mix, and transfer 100 mL aliquot to 250 mL beaker. 
Add 50 mL 0.1/V NaOH to beaker, stir well, let stand 30 min at 
room temp., and back-titr. with 0.1/V HCI, using pH meter (or 
indicator, 6.420(a), if pH meter is not available). Calc. % tetra- 
ethylpyrophosphate = net mL 0.1/V NaOH x 3.67/wt sample 
taken. 

(b) In formulations of tetraethylpyrophosphate containing 
organic solvent and emulsifying agent. — Proceed as in (a), 
except filter acetone soln thru 25 mm cotton plug in cylindrical 



• Surplus method — see inside front cover. 



funnel (25 mm diam., 75 mm long) before adding it to column 
if oil seps from soln. Pass acetone washings successively thru 
separator, cylindrical funnel, and resin column as in (a). (Cotton 
plug absorbs oil.) 



Organic Thiocyanates 

Thiocyanate Nitrogen in Livestock or 
Fly Sprays (106) — Official Final Action 

{Caution: See 51.041.) 

6.423 Reagents 

(a) Strong potassium polysulfide soln. — Dissolve 180 g KOH 
in 120 mL H 2 0. Sat. 100 mL of this soln with H 2 S (ca 42 g) 
(Caution: See 51.059) while cooling. Add remaining 100 mL KOH 
soln and 80 g S. Shake until dissolved. 

(b) Mixed sulfide soln.— To 100 mL (a) add 50 g Na 2 S.9H 2 0, 
30 g KOH, and 200 mL H 2 0. 

(c) Sodium bisulfite.— Na 2 S 2 5 or NaHS0 3 . 

(d) Copper sulfate soln— -20% aq. soln CuS0 4 .5H 2 0. 

(e) Wash soln.— To 300 mL H 2 add 1 mL H 2 S0 4 (1+4), 1 g 
(c), 10 mL (d), and 12 g Na 2 S0 4 , and pass S0 2 into soln 10 min. 



6.424 



Preparation of Sample 



Weigh sample preferably contg ca 0.03 g thiocyanate N into 
250 mL g-s erlenmeyer. (If SCN content is very low, do not 
unduly increase amt sample without correspondingly increasing 
amt mixed sulfide soln used; 20-25 g fly spray is usually 
enough.) Add 35 mL mixed sulfide soln and shake vigorously 
at room temp. 10 min, during which time reaction is nearly 
completed. Heat to 70° on steam bath, carefully releasing 
pressure resulting from heating, shake 15 min at 70°, and cool. 

Removal of petroleum oil .—Transfer mixt to separator with 
ca 200 mL H 2 0. Add 50 mL pet ether, shake, and drain aq. layer 
into 600 mL beaker. Wash pet ether layer with two 10 mL 
portions H 2 0, adding washings to main soln. (If emulsions form 
during washing, break by acidifying with H 2 S0 4 (1+4).) Drain 
aq. layer and wash pet ether layer with H 2 as above. Discard 
pet ether layer. 



6.425 



Determination 



Dil. combined aq. soln to ca 300 mL and neutze with H 2 S0 4 
(1+4), using litmus paper as outside indicator. Add 2 mL H 2 S0 4 
(1+4), quickly bring mixt. to bp, and boil 8 min to remove H 2 S. 
Cool. If fatty acids or oils are present, transfer to separator, ext 
with pet ether, and return aq. phase to original beaker. Filter 
thru small buchner and transfer filtrate to beaker. Neutze to 
litmus paper with 10% KOH soln and add 1 mL H 2 S0 4 (1+4). 
Add 1 g Na bisulfite and stir until dissolved. Add excess (ca 15 
mL) CuS0 4 soln and pass S0 2 into soln 10 min. 

Let pptd CuSCN settle 2 hr, and filter with suction thru 56 mm 
buchner coated with layer of asbestos (Caution: See 51.086), 
upon which is placed No. 42 Whatman paper, or equiv., second 
layer of asbestos, layer of diatomite, and finally third layer of 
asbestos. If filtrate is not clear, centrf. soln at 2000 rpm 10-15 
min, and pour thru filter again. Wash filter and ppt once or twice 
with wash soln, continue suction until filter pad is dry, and 
transfer to 800 mL Kjeldahl flask. (Filter pad may be folded in 
filter paper together with bits of moist filter paper used to wipe 
out buchner, and whole placed in Kjeldahl flask.) Add few glass 
beads, 35 mL H 2 S0 4 , 10 g K 2 S0 4 , and ca 0.7 g HgO or 0.65 g Hg. 
(Caution: See 51.030 and 51.065.) Digest until colorless; then 15 
min more. Det. N as in 2.057, second par. Perform blank analysis 
on paper, filter pad, and reagents. 



AOAC Methods (1980) 



Triazines 



121 



Thiocarbamate Herbicides (107) — Official First Action 

(S-Ethyl Dipropylthiocarbamate (EPTC, Eptam®), S-Ethyi 

Hexahydro-1H-azepine-1-carbothioate (Molinate, Ordram®), 

S-Ethyl Cyclohexylethylthiocarbamate (Cycloate, Ro-Neet®), 

S-Ethyl Diisobutylthiocarbamate (Butylate, Sutan®}, 

S-Propyl Butylethylthiocarbamate (Pebulate, Tillam®), 

S-Propyl Dipropylthiocarbamate 

(Vernolate, Vernam®)} 

Gas Chromatographic Method 

(Applicable to liq. and granular formulations. 
Caution: See 51.041.) 



6.426 



Apparatus 



(a) Gas chromatography — With flame ionization detector. Op- 
erating conditions: temps (°) — injection port 225, column 130 
(EPTC and butylate), 170 (molinate), 140 (cycloate, vernolate), 
150 (pebulate), detector 250; gas flows (mL/min)— N carrier 
30-35, H 25-30, air 200-300 (or as specified by manufacturer). 

(b) Recorder. — 1 mv full scale sensitivity and 1 sec response. 

(c) Columns,— 6' (1.8 m) x 0.25" od, Pyrex, Al, or stainless 
steel, packed with 3% OV-17 on 60-80 Gas-Chrom Q, or equiv. 
(for molinate), and 3% SE-30 or OV-1 on 60-80 mesh Gas-Chrom 
Q, or equiv. (for other 5 compds). Condition columns 12 hr at 
250° under N flow of 30 mL/min. 

6.427 Preparation of Standards 

(a) Internal std soins. — Accurately weigh ca 400 mg each ref. 
grade thiocarbamate (EPTC, cycloate, butylate, or pebulate; 
Stauffer Chemical Co., 1200 S 47th St, Richmond, CA 94804) 
and transfer to sep. 100 mL vol. flasks. Dil. to vol. with CS 2 - 
CHCI 3 -MeOH (80+15+5), and mix thoroly. 

(b) Std solns. — Accurately weigh ca 100 mg each ref. grade 
thiocarbamate into sep. 2 oz (50 mL) polyethylene-lined screw- 
cap, conical bottles. Add 25 mL internal std soln indicated below, 
and mix thoroly. 





Approx. 




Approx. 




retention 




retention 




time. 


Internal std 


time, 


Std soln 


min 


soln added 


min 


EPTC 


2.0 


Butylate 


2.4 


Molinate 


4.3 


Cycloate 


4.8 


Cycloate 


5.4 


Pebulate 


2.6 


Butylate 


2.4 


EPTC 


2.0 


Pebulate 


4.0 


Cycloate 


8.0 


Vernolate 


3.5 


Cycloate 


5.5 



6.428 Preparation of Sample 

Accurately weigh sample contg ca 100 mg thiocarbamate into 
2 oz (50 mL) polyethylene-lined screw-cap, conical bottle. Add 
25 mL appropriate internal std soln, (a), as indicated in (b), and 
shake thoroly. Vigorously shake granular formulations 30 min 
on wrist-action shaker. 



6.429 



Determination 



Inject 2 jitL clear supernate or soln into chromatograph pread- 
justed to appropriate conditions. Make triplicate injections of 
sample and appropriate std soln in random order. Det. peak 
areas, preferably with digital integrator. 

Adjust sensitivity of gas chromatograph so that larger com- 
ponent or internal std peak is ca % full scale. 



6.430 Calculations 

Response Factor (RF) = (/' x g compd in std soln x % purity)/S' 

Wt % compd = (RF x S)/l x g sample), 
where / and /' = areas of internal std peak in sample and std 
solns, resp,; and S and S' = areas of compd peak in sample and 
std solns, resp. 

Triazines and Other Pesticides (108) — Official Final Action 

(See Table 6:03 for applicability to and official status of specific 
compds.) 



AOAC-CiPAC Method 



6.431 



Standard Solutions 



[Caution: See 51.041.) 

(a) Dieldrin internal std soln. — Std should be 3=90% pure and 
contain no impurities eluting at retention time for pesticide 
being detd. (1) For propazine. — Weigh 14.0 ±0.1 g tech. dieldrin, 
dissolve in ca 300 mL CHCI 3 , and dil. to 1 L with CHCl 3 . (2) For 
other compounds. — Weigh 2.00±0.02 g tech. dieldrin, dissolve 
in ca 200 mL CHCI 3 , and dil. to 250 mL with CHCI 3 . 

(b) Aidrin internal std soln.— {For Diazinon®.) Weigh 4.0±0.1 
g tech. aldrin into 600 mL beaker. Slurry with 400 mL acetone 
to dissolve, filter thru paper into 1 L vol. flask, washing with 
several 100 mL portions acetone, and dil. to vol. Std should be 
3=90% pure and contain no impurities eluting at retention time 
of Diazinon. 

(c) Di benzyl succinate internal std soln. — (For chlorobenzilate 
and chloropropylate.) Weigh 5.0±0.1 g dibenzyl succinate, dis- 
solve in ca 300 mL acetone, and dil. to 1 L with acetone. Std 
should be >98% pure and contain no impurities eluting at 
retention time for pesticide being detd. 

(d) Pesticide std solns. — Accurately weigh 250 mg (125 mg 
for Diazinon and 150 mg for simazine) of ref. std of pesticide 
being detd (available from Ciba-Geigy Corp., PO Box 11422, 
Greensboro, NC 27409) into 4 oz (125 mL) round bottle with Al- 
lined screw cap. Pipet in 50 mL internal std soln {see Table 6:03) 
and shake mech. 30 min. 

(e) Dioctyl phthalate internal std soln. — (For simazine.) Weigh 
3.0±0.1 g tech. dioctyl phthalate, dissolve in ca 200 mL DMF, 
and dil. to 1 L with DMF. {Caution: See 51.053.) Std should be 
>98% pure and contain no impurities eluting at retention time 
of simazine. 



6.432 



Preparation Of Sample 



Accurately weigh amt sample specified in Table 6:03 into 4 
oz (125 mL) round bottle with Al-lined screw cap. Pipet in same 
vol. internal std used for prepn of std soln, (d), and shake mech. 
30 min. Let insol. materials settle or centrf. portion of ext to 
obtain clear soln. 



6.433 



Gas Chromatography 



Use instrument equipped with flame ionization detector and 
4 mm id glass column (length specified in Table 6:03) packed 
with 3% Carbowax 20M (Applied Science Laboratories, Inc.) on 
80-100 mesh Gas-Chrom Q. (For Diazinon, use 10% silicone DC- 
200 viscosity 12500.) Condition 24 hr at 240° with N or He at ca 
40 mL/min. Column should have 5=2000 (5=1500 for chloroben- 
zilate, chloropropylate, propazine, and simazine) theoretical 
plates (see 6.156(a)). 

Operate at following conditions: temps — as specified in Table 
6:03; N or He carrier gas, 80-100 mL/min; air and H, 80-100 
mL/min; attenuation varied so that peak hts of pesticide and 
internal std are 60-80% fulj scale. Retention times are specified 



122 



6. Pesticide Formulations 



AOAC Methods (1980) 



Table 6:03 Chemical and Gas Chromatographic Parameters for Triazines and Other Pesticides 



Chemical Name 



CA Internal Length 

Common or Registry Std Soln Column 

Trade Name No. 6.431 Wt Sample m 



Temperature (°) 
Inlet Column Detector 



Retention Times (min) 
Pesticide Internal Std 



2-(Ethylamino)-4-(isopropylamino)- 
6-(methylthio)-s -triazine 

2-Chforo-4-(ethylamino)-6- 
(isopropylamino)-s-triazine 

Ethyl-4,4'-dichlorobenzi!ate 
lsopropyl-4,4'-dichlorobenzilate 

0,0-Diethyl-0-(2-isopropyl-6- 

methyl-4-pyrimidinyl) 

phosphorothioate 
2,4-Bis(isopropylamino)-6- 

methoxy-s-triazine 
2,4-Bis(isopropylamino)-6- 

(methylthio)-s-triazine 

2-Chloro-4,6-bis(isopropylamino)- 
s-triaztne 

2-Chloro-4,6-bis(ethylamino)-s- 
triazine 

2-(te/t-Butylamino)-4-(ethylamino)- 
6-{methylthio)-s -triazine 



Ametryn 834-12-8 

Atrazine 1912-24-9 

Chlorobenzilate 510-15-6 

Chloropropylate 5836-10-2 

Diazinon 333-41-5 

Prometon 1610-18-0 
Prometryn 7287-19-6 

Propazine 139-40-2 

Simazine 122-34-9 

Terbutryn 886-50-0 



(a)(2) 


300 mg 80% 
wettable 
powder 


1.8 


240 


215±15 


240 


(a)(2) 


300 mg 80% 
wettable 
powder 


1.8 


240 


200±10 


240 


(0 


500 mg liq. 
formulation 


1.2 


260 


230±10 


260 


(c) 


1 g liq. 
formulation 


1.2 


260 


230±10 


260 


(b) 


Sample 

contg 110 

mg 


1.8 


240 


190±10 


240 


(a)(2) 


1 g Mq. 

formulation 


1.8 


240 


200±20 


240 


(a)(2) 


300 mg 80% 
wettable 
powder 


1.8 


240 


200±10 


240 


(a)(7) 


300 mg 80% 
wettable 
powder 


.2 


250 


210±10 


240 


(e) 


190 mg 80% 
wettable 
powder 


1.8 


250 


210±5 


250 


(a)(2) 


300 mg 80% 
wettable 
powder 


.8 


240 


200±20 


240 



8-12 



5-7 



3-5 



6-8 



8-10 



9-15 



9-15 



5-8 


8-10 


4-6 


8-10 


5-6 


10-12 


3-5 


9-15 


6-8 


9-15 



7-9 



10-14 



9-15 



in Table 6:03. (Ametryn and dieldrin peaks must be resolved. 
Prep, new column if variation of flow rate or temp, does not 
resolve peaks. Resolution may be improved by increasing 
column temp.) 



6.434 



Determination 



Inject 3 jliL aliquots std soln until peak ht ratio of pesticide: 
internal std varies ^1% for successive injections. Then make 
duplicate injections of sample followed by duplicate injections 
of std. Peak ht ratios of stds must be within ± 1 % of first accepted 
std values or repeat series of injections. Repeat for addnl 
samples. 

6.435 Calculations 

Calc. peak ht ratios for both duplicate std injections preceding 
and following samples. Average the 4 values (/?'). Calc. and 
average peak ht ratios of the 2 samples (/?). 

% Pesticide = <fl/W) x (W x P/R'), 
where W and W = mg sample and std, resp.; andP = % purity 
of std. 



Triphenyltin Compounds (Fentin) 

Potentiometric Titration Method {109)— Official First Action 
{CIPAC Method) 

6.436 Principle 

Org. Sn compds are extd with acetone, diphenyltin compds 
are quant, converted to msol. oxide with alk. alumina and 
filtered, and aceione soln is titrd potentiometrically. 



6.437 



Apparatus 



(a) Filtration apparatus. — Glass bell with neck and removable 
plate to permit glass buchner with fine porosity fritted disk and 
long stem to drain into beaker under vac. (Fig. 6:09). 



(b) Potentiometric titration apparatus. — pH meter with glass 
and satd calomel electrodes is satisfactory. 

6.438 Reagents 

{a) Alkaline alumina. — Mix 150 g neutral Al 2 3 (Woelm 4649, 
or equiv.) with 150 mL alcohol contg 15 g KOH in 1 L r-b flask. 
Reflux 30 min, cool, and filter with suction thru buchner. Dry 
powder in vac. 1 hr at 100° and 3-3.5 hr at 130°. Pour warm 
powder into bottle and stopper tightly. Com. alk. Al 2 3 is not 
satisfactory. 

(b) Cellulose powder. — Whatman CF 11, or equiv. 




imimimiHiiwimimiH 



FIG. 6:09 — Filtration apparatus 



AOAC Methods (1980) 



Selected References 



123 



6.439 



Determination 



Accurately weigh into 100 mL glass beaker sample contg ca 
0.30 g triphenyltin compd. Add 2 g alk. Al 2 3 and 25 mL acetone 
and stir with mag. stirrer 10 min. Prep, and process blank of 2 
g alk. Al 2 3 and 25 mL acetone in same manner. Place 1 g 
cellulose powder and 1 g alk. Al 2 3 in funnel and mix thoroly. 
Assemble filtration app. contg 250 mL beaker and filter suspen- 
sion thru funnel. Wash beaker and funnel with four 20 mL 
portions acetone. Titr. filtrate potentiometrically with stdzd 0.1/V 
HCI. 

% Triphenyltin compd = (S - B) x N x {M/W) x 10 
where S = mL HCI used for sample, B = mL HCI used for blank, 
N = normality of HCI, M = mol. wt of compd (367.0 for 
triphenyltin hydroxide and 409.0 for the acetate), and W - g 
sample. 



QUATERNARY AMMONIUM COMPOUNDS 

Chloride (710)— Official Final Action 

6.440 Potentio metric Titration Method 

Transfer sample contg 30-35 mg CI to 600 mL beaker, dil. to 
200 mL with H 2 0, and add 5 mL HN0 3 (1 + 1). Add just enough 
acetone to dissolve ppt that forms and titr. with 0.1 N AgN0 3 , 
using app. for potentiometric titrn. Calc. % CI (1 mL 0.1/V AgN0 3 
= 3.545 mg CI) and equiv. % quaternary NH 4 salt. 



Adsorption indicator Method 



6.441 



Reagents 



(a) Bromothymoi blue indicator. — Dissolve 1 g indicator in 
500 mL 50% alcohol. 

(b) Dichlorofluorescein soln. — 0.1%, Dissolve 100 mg indi- 
cator in 100 mL 70% alcohol. 



6.442 



Determination 



Transfer sample contg 30-140 mg CI (usually ca 1 g quaternary 
NH 4 salt) into 300 mL erlenmeyer, dil. to 75 mL with H 2 0, and 
add 25 mL isopropanol. Neutze if necessary with HOAc (1+9), 
using 1 drop bromothymoi blue (pH 4-6). Add 10 drops dich- 
lorofluorescein, and titr. with 0.1/V AgN0 3 , avoiding direct sun- 
light. Ppt becomes red at end point and may flocculate just 
before end point. Calc. % CI and equiv. % quaternary NH 4 salt. 



SELECTED REFERENCES 

(7) J AOAC 62, 494(1979) 

(2) Ind. Eng. Chem. 14, 207(1922); JAOAC 5, 33, 402(1922); 
6,313(1923). 

(3) JAOAC 7, 313(1924). 

(4) Anal. Chem. 22, 1066(1950); JAOAC 46, 672 (1963). 

(5) JAOAC 5, 398(1922). 

(6) J. Am. Chem. Soc. 40, 1036(1918); JAOAC 5, 398(1922). 

(7) J. Res. Natl. Bur. Standards 3, 581(1929); JAOAC 25, 670 
(1942); 27, 74(1944); 28, 72(1945). 

(8) Compt. rend. 173, 714, 836(1921); JAOAC 14, 253(1931). 

(9) J. Am. Chem. Soc. 55, 1741(1933); Ind. Eng. Chem., Anal. 
Ed. 5, 7(1933); 9, 551(1937); 11, 21(1939); JAOAC 21, 
459(1938). 

(70) JAOAC 55, 851(1972). 
(7 7) JAOAC 43, 367(1960). 
(12) Ind. Eng. Chem. 1, 208(1909); JAOAC 3, 158(1917). 



(73 
(74 

(75; 

(16] 
(17 

(18] 
(19] 

{2o: 

(21 



(22 
(23 
(24 
(25] 
(26] 
(27] 
(28] 
(29] 
(30] 
(31 
(32 
(33 
(34] 
(35] 
(36] 
(37] 

(38] 
(39] 
(40] 
(41 
(42 
(43] 
(44] 
(45] 
(46] 

(47] 
(48] 
(49 

(50] 

(51 
(52 
[53] 
(54] 
(55] 
(56] 
(57] 



(58] 
(59] 
(60] 
(61 
(62 
(63 
(64] 
(65] 
(66] 
(67] 
(68] 
(69] 
(70 



JAOAC 5, 34(1922). 

J. Am. Chem. Soc. 24, 1082(1902). 

USDA Bur. Chem. Bull. 105, p. 167. 

JAOAC 3, 332(1920). 

JAOAC 3, 333(1920). 

USDA Bur. Chem. Bull. 137, p. 40; 152, p. 68. 

JAOAC 5, 33(1921); 392(1922). 

JAOAC 47, 253(1964). 

Fresenius, "Quantitative Chemical Analysis/' Trans. 6th 

German Ed., 1906, amplified and revised, Vol. 2, 1180; 

U.S. Geol. Survey Bull. 700, p. 218. 

JAOAC 15, 289(1932); 17, 62(1934). 

JAOAC 10, 29(1927). 

JAOAC 35, 377(1952). 

JAOAC 10,27(1927) 

JAOAC 10,28(1927). 

JAOAC 3, 353(1920). 

JAOAC 18, 63, 65(1935); 43, 346(1960). 

JAOAC 43, 346(1960). 

JAOAC 10, 30, 124(1927); 11, 35(1928). 

USDA Bull. 898, p. 48. 

JAOAC 9, 27(1926). 

USDA Bur. Chem. Circ. 10, p. 7. 

JAOAC 9, 28(1926). 

USDA Bur. Chem. Bull. 105, p. 165. 

JAOAC 9, 29(1926). 

Whitmore, "Organic Compounds of Mercury," p. 365; 

JAOAC 13, 156(1930). 

JAOAC 54, 685(1971). 

JAOAC 56, 572(1973). 

JAOAC 31, 366(1948). 

JAOAC 22, 411(1939); 25, 79(1942); 28, 72(1945). 

JAOAC 43, 365(1960). 

JAOAC 48, 576(1965); 49, 207(1966). 

Anal. Chem. 25, 1207(1953); JAOAC 40, 732 (1957). 

JAOAC 55, 907(1972). 

Ind. Eng. Chem., Anal. Ed. 10, 19(1938); JAOAC 21, 

148(1938); 22, 408(1939); 24, 70(1941); 43, 376(1960). 

JAOAC 44, 580(1961); 46, 668(1963). 

JAOAC 35, 771(1952); 43, 350(1960). 

Contrib. Boyce Thompson Inst. 8, No. 3, 175(1936); Ind. 

Eng. Chem., Anal. Ed. 10, 5(1938); JAOAC 43, 358(1960). 

Soap 10, No. 5, 89(1934); JAOAC 43, 354(1960); 46, 

664(1963). 

JAOAC 43, 374(1960). 

USDA Bur. Animal ind. Bull. 133. 

JAOAC 44, 595(1961). 

JAOAC 53, 1155(1970). 

JAOAC 32, 684(1949); 39, 373(1956). 

Ind. Eng. Chem. 8, 312(1936). 

Anal. Chem. 19, 779(1947); Report No. 4760, May 15, 1949, 

Phys. Chem. Lab., Hooker Electrochemical Co., Niagara 

Falls, NY. 

Anal. Chem. 25, 1661(1953); JAOAC 40, 737(1957). 

JAOAC 56, 567(1973). 

JAOAC 54, 688(1971). 

JAOAC 45, 513(1962). 

JAOAC 48, 573(1965). 

JAOAC 49, 254(1966). 

JAOAC 55, 942(1972). 

JAOAC 59, 716(1976); 61, 1499(1978). 

JAOAC 62, 8(1979). 

JAOAC 30, 319(1947); 31, 368(1948). 

JAOAC 40, 286(1957); 43, 342(1960). 

JAOAC 52, 1284(1969). 

JAOAC 51, 1301(1968). 



124 



6. Pesticide Formulations 



AOAC Methods (1980) 



(77) JAOAC 54, 706(1971), 

(72) JAOAC 61, 1163(1978). 

(73) Anal. Chem. 31, 418(1959); JAOAC 43, 382 (1960); 45, 
522(1962). 

1109(1976). 

716(1977). 

1157(1977). 

565(1968). 

748(1976). 

1774(1955); JAOAC 43, 382(1960); 45, 



(74) 
(75) 
(76) 
(77) 
(78) 
(79) 

(80) 
(81) 
(82) 
(83) 
(84) 
(85) 

(86) 
(87) 



JAOAC 59, 

JAOAC 60, 

JAOAC 60, 

JAOAC 51, 

JAOAC 59, 

Anal. Chem. 27, 

522(1962). 

JAOAC 57, 642(1974). 

JAOAC 50, 568(1967). 

JAOAC 50, 

JAOAC 47, 

JAOAC 49, 

Analyst 92, 

857(1972). 

JAOAC 48, 562(1965). 

J. Sci. Food Agric. 15, 509(1964); JAOAC 49, 40(1966); 

447(1968). 

(88) JAOAC 52, 1292(1969). 

(89) Ber. 31, 2979(1898); J. Am. Chem. Soc. 27, 1183(1905); 
USDA Bur. Chem. Bull. 99, p. 30; 132, p. 49; 137, p. 47. 



566(1967); 56, 576(1973); 59, 753, 1196(1976). 

268(1964). 

251(1966). 

375(1967); JAOAC 51, 1304, 1306 (1968); 55, 



51, 



(90) Z. anal. Chem. 36, 18(1897); USDA Bur. Chem. Bull. 132, 
p. 49. 

(91) Ind. Eng. Chem., Anal. Ed. 3, 357(1931); JAOAC 25, 80, 
668(1942). 

(92) JAOAC 62, 11(1979). 

(93) JAOAC 57, 771(1974). 

(94) JAOAC 55, 1133(1972). 

(95) JAOAC 55, 926(1972). 

(96) JAOAC 62, 272(1979). 

(97) JAOAC 61,495(1978). 

(98) Anal. Chem. 23, 1167(1951); JAOAC 35, 381(1952); 36, 
384(1953). 

(99) JAOAC 43, 344(1960); 47, 242(1964). 

(100) JAOAC 60, 720(1977). 

(101) JAOAC 60, 724(1977). 
(702) JAOAC 60, 862(1977). 

(103) JAOAC 47, 245(1964). 

(104) JAOAC 51, 562(1968). 
(705) Anal. Chem. 21, 808(1949). 
(106) JAOAC 34, 677(1951). 
(707) JAOAC 57, 53(1974). 

(108) JAOAC 56, 586(1973); 58, 513, 516(1975); 59, 758(1976). 
(705) JAOAC 61, 1504(1978). 
(7 70) JAOAC 43, 352(1960). 



7. Animal Feed 



7.001 Sampling {/) — Procedure 

Use slotted single or double tube, or slotted tube and rod, all 
with pointed ends. 

Take 5=500 g sample, 1 kg preferred, as follows: Lay bag 
horizontally and remove core diagonally from end to end. Det. 
number of cores as follows: From lots of 1-10 bags, sample all 
bags; from lot of 5=11, sample 10 bags. Take 1 core from each 
bag sampled, except that for lots of 1-4 bags take enough 
diagonal cores from each bag to total ^5 cores. For bulk feeds 
draw 5=10 cores from different regions; in sampling small 
containers (^10 lb) 1 package is enough. Reduce composite 
sample to amt required, preferably by riffling, or by mixing 
thoroly on clean oil-cloth or paper and quartering. Place sample 
in air-tight container. 

A sample from less than these numbers of bags may be 
declared an official sample if guarantor agrees. For samples that 
cannot be representatively taken with probe described, use 
other sampling means. 

7.002 Preparation of Sample — Official Final Action 

Grind sample to pass sieve with circular openings 1 mm C/25") 
diam. and mix thoroly. If sample cannot be ground, reduce to 
as fine condition as possible. Do not grind molasses feeds. 

Moisture — Official Final Action 
/. Drying in Vacuo at 95-100? (2) 



7.003 



Determination 



Dry amt sample contg ca 2 g dry material to const wt at 
95-100° under pressure ^100 mm Hg (ca 5 hr). For feeds with 
high molasses content, use temp. =s70° and pressure =s50 mm 
Hg. Use covered Al dish 5=50 mm diam. and =s40 mm deep. 
Report loss in wt as moisture. 



//. By Distiilation with Toluene (3) 



7.004 



Apparatus 

Connect 250 ml_ flask of Pyrex or other resistant glass by 
means of Bidwell-Sterling moisture receiver to 500 mm Liebig 
condenser. Calibrate receiver, 5 ml_ capacity, by distg known 
amts H 2 into graduated column, and estg column of H 2 to 
0.01 mL. Clean tube and condenser with chromic acid cleaning 
mixt., rinse thoroly with H 2 0, then alcohol, and dry in oven to 
prevent undue amt H 2 from adhering to inner surfaces during 
detn. 



7.005 



Determination 



If sample is likely to bump, add dry sand to cover bottom of 
flask. Add enough toluene to cover sample completely (ca 75 
mL). Weigh and introduce enough sample into toluene to give 
2-5 mL H 2 and connect app. Fill receiving tube with toluene, 
pouring it thru top of condenser. Bring to boil and distil slowly, 
ca 2 drops/sec, until most of the H 2 passes over; then increase 
rate of distn to ca 4 drops/sec. 

When all H 2 is apparently over, wash down condenser by 
pouring toluene in at top, continuing distn short time to see 
whether any more H 2 distils over; if it does, repeat washing- 
down process. If any H 2 remains in condenser, remove by 



brushing down with tube brush attached to Cu wire and satd 
with toluene, washing down condenser at same time. (Entire 
process is usually completed within 1 hr.) Let receiving tube 
come to room temp. If any drops adhere to sides of tube, force 
them down, using Cu wire with end wrapped with rubber band. 
Read vol. H 2 and calc. to %. 

7.006 it HI. Drying without Heat over it 

Sulfuric Acid (4) 

See 7.006-7.007, 12th ed. 

7.007 IV. Drying at 135° (5) 

(Not to be used when fat detn is to be made on same sample) 

Regulate air oven to 135±2°. Using low, covered Al dishes, 
7.003, weigh ca 2 g sample into each dish and shake until 
contents are evenly distributed. With covers removed, place 
dishes and covers in oven as quickly as possible and dry samples 
2 hr. Place covers on dishes and transfer to desiccator to cool. 
Weigh, and calc. loss in wt as H 2 0. 

7.008 V. In Highly Acid Milk By-products [6) 

Add ca 2 g ZnO, freshly ignited or oven dried, to flat-bottom 
dish 5=5 cm diam. and weigh. Add ca 1 g sample and weigh 
quickly. Add ca 5 mL H 2 and distribute sample evenly on 
bottom of dish. Heat on steam bath, exposing max. surface of 
dish bottom to live steam until apparently dry. Heat at 98-100° 
in air oven 3 hr or to const wt. Cool in desiccator; then weigh 
quickly. Det. wt residue. Titr. acidity of sample and calc. as lactic 
acid, 16.023. To compensate for H 2 formed when acid is neutzd 
by ZnO, add 0.1 g to residue wt for each g acid (as lactic) in 
weighed sample. Report % residue (corrected) as total solids. 

7.009 Ash (7)— Official Final Action 

Weigh 2 g sample into porcelain crucible and place in temp. 
controlled furnace preheated to 600°. Hold at this temp. 2 hr. 
Transfer crucible directly to desiccator, cool, and weigh imme- 
diately, reporting % ash to first decimal place. 



NITROGEN 
Qualitative Tests for Proteins [8)— Official Final Action 

7.010 it Biuret Test it 
See 22.012-22.013, 10th ed. 

7.011 it MillonTest • 

See 22.014-22.015, 10th ed. [Caution: See 51.018, 51.026, and 
51.065.) 

7.012 it Glyoxylic Acid Test {Hopkins-Cole) it 

See 22.016-22.017, 10th ed. [Caution: Wear face shield and 
heavy rubber gloves as protection against reagent bump. See 
also 51.018 and 51.063.) 



* Surplus method — see inside front cover. 



125 



126 



7. Animal Feed 



AOAC Methods (1980) 



7.013 * Adamkiewicz Test * 
See 22.018, 10th ed. 

7.014 it Xanthoproteic Test • 
See 22.019, 10th ed. 

Crude Protein — Official Final Action 

7.015 Kjeldahl Method (9) 

Det. N as in 2.057. Multiply result by 6.25, or in case of wheat 
grains by 5.70. 



Dumas Method [10) 



7.016 



Principle 

N, freed by pyrolysis and subsequent combustions, is swept 
by C0 2 carrier into nitrometer. C0 2 is absorbed in KOH and vol. 
residual N is measured and converted to equiv. protein by 
numerical factor. 



7.017 



Apparatus and Reagents 



(a) Nitrogen analyzer and accessories . — Consists of combus- 
tion and collection and measuring systems. Suitable instrument, 
Model 29A, with following accessories and reagents is available 
from Coleman Instruments Div., 2000 York Rd, Oak Brook, IL 
60521: Al combustion boats, No. 2&-412; Vycor combustion 
tubes, No. 29-328; CuO-Pt catalyst {CuO wire form with 2.5% 
Pt reforming catalyst), No. 29-160; reduced Cu wire, No. 29-120; 
Co 3 4 , No. 29-170; CuO powder, fines, No. 2&-140; 45% KOH, 
No. 29-110. 

(b) Balance. — Accurate to 0.01 mg. 

(c) Barometer. — Hg type, readable to 0.1 mm. 

7.018 Preparation of Samples 

Grind to pass No. 30 sieve. Store in capped bottles. 



7.019 



Determination 



Operate instrument in accordance with instructions of man- 
ufacturer. (Following directions apply to Coleman Model 29A 
Nitrogen Analyzer. Consult Operating Directions D-360B, Cole- 
man Cat. No. 29-904, for addnl details.) 

After combustion furnaces have come to thermal equilibrium, 
turn combustion cycle control to START and let proceed normally 
thru cycle. Observe indicated temp, on pyrometer of both upper 
and lower combustion furnaces at end of combustion portion 
of cycle. Furnace temps should be 850-900°. If not, adjust. 

Prep, combustion tube by inserting stainless steel screen in 
lower end of combustion tube (end farthest from trademark). In 
upper end, place enough glass wool to form 6 mm plug when 
packed. With 11 mm glass rod, drive glass wool down to 
stainless steel plug. Holding tube vertically, pour CuO-Pt catalyst 
directly from dispenser bottle into combustion tube until it 
reaches upper end of trademark. Tap or vibrate tube on bench 
until reagent settles to approx. center of trademark. 

Weigh and record wt of empty Al combustion boat. Place 
sample in boat. Weigh and record wt of sample and combustion 
boat. Difference between wts is sample wt. Use following sample 
wts (mg) as guides to suitable sample sizes: bermuda grass 
150-300; rice bran, wheat shorts, dehydrated alfalfa 150-250; 
range feed 100-200; cottonseed meal 75-150; edible soy protein 
50-150. Weigh sample to nearest 0.01 mg. To avoid wt changes, 

-A- Surplus method — see inside front cover. 



Table 7:01 Volume correction for temperature correction 
factor (C f ) (AtL/°K) a 



Final Counter 

Reading 

(ML) 



(C f ) 

(Nitrometers with 

Check Value) 




5000 
10000 
15000 
200,00 
25000 
30000 
35000 
40000 
45000 
50000 



12 

29 

45 

62 

79 

95 

112 

129 

145 

162 

179 



a Vol. correction, V t = C f (t 2 - t,) 

record wt within 1 min after sample and boat are placed on 
balance. If this is impossible, weigh sample inside weighing 
bottle, such as Kimble No. 15165 or 15166. 

Turn combustion tube to horizontal, and carefully insert loaded 
sample boat into open end of tube. Slide or push boat, without 
spilling contents, until it reaches trademark. Raise open end 
until tube forms 60-70° angle to horizontal. Tap or vibrate 
combustion tube on bench top while rotating tube between 
thumb and forefinger. Raise open end of tube and add vol. 
CO3O4 and vol. CuO fines equal to vol. sample. For convenient 
means of adding above reagents to samples, place vol. CuO 
fines and vol. Co 3 4 , each equal to vol. sample, in addnl 
combustion boat; add contents of boat, but not boat itself, to 
combustion tube; and rotate partially filled combustion tube 
between thumb and forefinger while varying angle of tube 
20-45° from horizontal. Continue rotating, tapping, and vibrating 
until sample is dispelled from boat and is thoroly mixed with 
oxidizing agents. Raise open end until tube forms 60-70° angle 
to horizontal; add CuO-Pt catalyst ca 12 mm above sample boat. 
Tap or vibrate gently to eliminate voids. Add CuO-Pt catalyst to 
within 20 mm of top of tube, again tapping or vibrating gently 
to eliminate voids. 

Install prepd combustion tube in N analyzer. Adjust 45% KOH 
soln meniscus to calibrating mark in nitrometer with digital 
readout meter. Record counter reading, R,. (Counter reading 
should preferably lie between 500 and 1000 juL at this point. 
Vent control may be used to assist in arriving at this counter 
setting, if necessary.) Record syringe temp., t 1# indicated on 
special scale thermometer. Add 2 min more to combustion 
portion of cycle by turning auxiliary timer to setting 3. (Once 
this is done, addnl 2 min will be automatically programmed into 
each subsequent cycle.) Turn combustion cycle control to 
START. Let analyzer proceed thru its cycle. After cycle is 
complete and combustion cycle control has entered STAND-BY 
section, readjust KOH meniscus to calibration mark with digital 
readout counter. Record new counter reading, R 2r and syringe 
temp., t 2 . Det. blank for instrument under same conditions as 
actual analysis except omit sample. 

Table 7:02 Barometric temperature correction (P b ) 





°C 




P 


(mm 


Hg) 




Temperature, 


700-749 








750-780 


10 




1.2 








1.3 


15 




1.8 








1.9 


20 




2.3 








2.5 


25 




2.9 








3.1 


30 




3.5 








3.7 


35 




4.1 








4.3 



AOAC Methods (1980) 



Nitrogen 



127 



7.020 



Calculations 



(a) Record observed N vol., V = R 2 - R u where V - observed 
N vol. (mL)//?i = initial counter reading, and R 2 - final counter 
reading. 

(b) Det. corrected N vol. (in /xL), V c = V Q - {V b + V t ), where 
V b = vol. blank (/-iL), V x = vol. correction for temp. (yxL) — C f (t 2 
- tj. C f is obtained from Table 7:01 (based on final counter 
reading); t 2 and t, are in °K. 

(c) Det. corrected barometric pressure, P c - P - {P b + P v ), 
where P = observed barometric pressure (mm Hg), P b = 
barometric temp, correction (from Table 7:02), a ndf\, = pressure 
correction for vapor pressure of KOH soln (from Table 7:03). 

{Note: Empirical approximation of {P b + P v ) = 11.0 will be 
satisfactorily accurate for P between 740 and 780 mm Hg and 
syringe temp, between 298 and 305°K.) 

(d) Calc. % N = (P c x V c x 0.0449)/(7 r x W), where T = final 
syringe temp, in °K and W = sample wt in mg. 

Example: 

P - 750.1 mm Hg at 25°C; W - 148.91 mg 

Start Finish 

Counter readings, blank 500 /xL 524/aL 

Counter readings, sample 524 6955 

t, = 302.7°K, t 2 = 303.0°K, V = 6955 - 524 = 6431 juL 
V c = 6431 - [24 + C,(t 2 - tj] = 6431 - (24 + 35 x 0.3) - 6396 
fjil 
P c = 750.1 - (3.1 x 9.6) - 737.4 
% N - (737.4 x 6396 x 0.04493)/ (303.0 x 148.91) = 4.69% 

(e) Calc. % protein = % N x 6.25, or % N x 5.70 in case of 
wheat grains. 

Table 7:03 Pressure correction (f v ) for vapor pressure of 
KOH (for practical purposes, temp, of KOH is same as syringe) 



Temperature, °K 



P v (mm Hg) 



288 
293 
298 
303 
308 
313 



4.1 
5.7 
7.4 
9.6 
12.5 
16.5 



ac/'d— 96-98%. (2) Hydrogen peroxide.— 30-35%. (3) Ammo- 
nium sulfate std solns. — {a} Std soln I. — Dissolve 30.000±0.030 
g (NH 4 ) 2 S0 4 in H 2 and dil. to 1 L with H 2 0. (b) Std soln II.— 
Dissolve 0.750±0.001 g (NH 4 ) 2 S0 4 in H 2 and dil. to 1 L with 
H 2 0. {4) Mixed indicator soln. — Dissolve 1.000 g Me red and 
0.250 g methylene blue in alcohol and dil. to 1 L with alcohol. 
Dil. 10 mL this soln to 1 L with H 2 0. (5) Sodium hydroxide- 
sodium thiosulfate soln.—$0% NaOH-8% Na 2 S 2 3 .5H 2 0. (6) Di- 
lute sulfuric acid soln.— 0.6%. Dil. 30 mL 96-98% H 2 S0 4 to 5 L 
with H 2 0. 



7.024 



Determination 



{Caution: See 51.019, 51.030, 51.065, and 51.070.) 

Place 3 Kjel-tabs in special flask (500 mL of design compatible 
to Foss instrument) in position 1. Shift dispenser arm over flask 
and depress H 2 S0 4 lever, initiating simultaneous addn of 10 mL 
30-35% H 2 2 and 12-15 mL 96-98% H 2 S0 4 (depending on fat 
content of sample). To flask, add accurately weighed sample 
(ca 1.0 g if <45% protein, and ca 0.5 g if >45% protein) wrapped 
in weighing paper and close lid. Flask automatically rotates to 
position 2 where sample digests 3 min, and then to position 3 
for 3 min addnl digestion. In position 4, flask is cooled by 
centrifugal blower, lid opens automatically, and 140 mL H 2 is 
added automatically. Flask rotates to position 5, where NaOH- 
Na 2 S 2 3 soln is automatically introduced in excess. Released 
NH 3 is steam distd quant, into 200 mL tall-form titrn beaker 
contg 50 mL mixed indicator soln, and is simultaneously titrd 
automatically with dil. H 2 S0 4 soln delivered by photometrically 
regulated syringe. Final position of syringe is measured by 
potentiometer, output of which feeds electronic circuitry for 
conversion to visual display and/or printout in % N or % protein 
with appropriate conversion factors. In position 6, flask is 
emptied. Calibrate instrument initially each day with aliquots of 
(NH 4 ) 2 S0 4 std solns and check periodically as stated in operating 
manual. 

Semiautomated Method ( 12)— Official Final Action 

7.025 Principle 

Samples are digested in 250 mL calibrated tubes, using block 
digestor. A of NH 3 -salicylate complex is read in flowcell at 660 
nm, or NH 3/ is distd into std acid and back-titrd with std alkali. 



Automated Method { 11) — Official Final Action 

7.021 Principle 

Automation of macro Kjeldahl method is in 6 steps: sample 
and reagent addn, initial and final digestion, cooling and diln, 
NaOH addn, steam distn and titrn, and automatic pumping of 
flask contents to waste. Chemistry is carried out in macro 
Kjeldahl flasks equipped with side arms which are rotated at 3 
min intervals thru each successive step. 

7.022 Apparatus 

(a) Kjeldahl {protein /nitrogen ) analyzer. — Kjel-Foss Auto- 
matic, Model 16210 (Foss America, Inc., PO Box 504, Rt 82, 
Fishkill, NY 12524), or equiv. 

(b) Weighing papers.— 120 x 120 mm N-free tissues, Foss 
America, Inc., or equiv. 

7.023 Reagents 

(a) Kjel-tabs. — Contg 5 g K 2 S0 4 and 0.25 g HgO (Foss America, 
Inc.). 

(b) Kjeldahl {protein /nitrogen) analyzer reagents. — Prep, fol- 
lowing according to manufacturer's instructions: (/) Sulfuric 



7.026 Apparatus 

(a) Block digestor. — Model BD-20 (Technicon Instruments 
Corp.) or Model DS-20 (Tecator, Inc., 1898 S Flatiron Ct, Boulder, 
CO 80301 ). Capable of maintaining 410° and digesting 20 samples 
at a time in 250 mL calibrated volumetric tubes constricted at 
top. Block must be equipped with removable shields to enclose 
exposed area of tubes completely at or above ht of constriction. 

(b) Automatic analyzer. — AutoAnalyzer with following mod- 
ules (Technicon Instruments Corp.), or equiv.: Sampler II or IV 
with 40/hr (2:1) cam (higher ratio cams result in carry-over and 
poorer peak sepn); proportioning pump III; NH 3 anal, cartridge 
No. 116-D531-01 (or construct equiv. manifold from flow dia- 
gram); AAII single channel colorimeter with 15 x 1.5-2.0 mm 
id tubular flowcell, matched 660 nm interference filters, and 
voltage stabilizer; and recorder of appropriate span. {See Fig. 
7:01.) 

7.027 Reagents 

(a) Phosphate-tartrate buffer soln. — pH 14.0. Dissolve 50 g 
NaK tartrate and 26.8 g Na 2 HP0 4 .7H 2 in 600 mL H 2 0. Add 
54 g NaOH and dissolve. Add 1 mL Brij-35 (Technicon Instru- 
ments Corp.), dil. to 1 L with H 2 0, and mix. 



128 



7. Animal Feed 



AOAC Methods (1980) 



(b) Sodium chloride-sulfuric acid soln. — Dissolve 200 g NaCI 
in H 2 in 2 L vol. flask. Add 15 ml_ H 2 S0 4 and 2 mL Brij-35. Dil. 
to vol. with H 2 and mix. 

(c) Sodium hypochlorite soln. — Dil. 6 mL com. bleach soln 
contg 5.25% available CI (Clorox, or equiv.) to 100 mL with H 2 
and mix, Prep, fresh daily. 

(d) Sodium nitroprusside-sodium salicylate soln. — Dissolve 
150 g NaC 7 H 5 3 and 0.3 g Na 2 Fe(CN) 5 .N0.2H 2 in 600 mL H 2 0. 
Add 1 mL Brij-35, dil. to 1 L with H 2 0, and mix. 

(e) Nitrogen std solns. — Prep. 6 stds by accurately weighing 
(±10 mg) 59, 118, 177, 236, 295, and 354 mg (NH 4 ) 2 S0 4 primary 
std (Fisher Scientific Co. No. A-938, or equiv.; dry 2 hr at 105° 
before use and assume theoretical value of 21.20% N after 
drying) into individual 250 mL digestion tubes. Proceed as in 
7.031, beginning "Add 9 g K 2 S0 4 , 0.42 g HgO, and 15 mL H 2 S0 4 
. . ." Stds may be stored and reused until exhausted. 

(f) Sodium hydroxide-potassium sulfide soln: — Dissolve 400 
g NaOH in H 2 0. While still warm, dissolve 30 g K 2 S in soln, and 
dil. to 1 L 

7.028 Analytical System 

If manifold is to be constructed, use clear std pump tubes for 
all air and soln flows. All fittings, coils, and glass transmission 
lines are AAII type and size. Use glass transmission tubing for 
all connections after pump to colorimeter. Construct modified 
AO fitting on sample diln loop using AO fitting, N13 stainless 
steel nipple connector, and Vz length of 0.035" id Tygon tubing. 
Insert N13 nipple approx. halfway into 0.035" Tygon tubing. 
Insert tubing into side arm of AO fitting far enough so resample 
line will not pump any air. Space pump tubes equally across 
pump rollers. Cut 0.16 mL/min resample pump tube *£1" at 
entrance before connecting to side arm of AO fitting. In opera- 
tion, add buffer and hypochlorite solns thru metal side arms of 
A10 type fittings; add salicylate soln, (d), thru metal insert to 
20T coil. Air, reagents, and sample are combined immediately 
after pump thru injection fittings. 



7.029 



Start-Up 



Start automatic system and place all lines except salicylate 
line in resp. solns. After ^5 min, place salicylate line in resp. 
soln and let system equilibrate. If ppt forms after addn of 
salicylate, pH is too low. Immediately stop proportioning pump 
and flush coils with H 2 0, using syringe. Before restarting system, 
check concns of NaCI-H 2 S0 4 soln and phosphate-tartrate buffer 
soln. 

Pump lowest concn N std soln continuously thru system 5=5 
min and adjust baseline control on colorimeter to read 10% full 
scale. Pump highest concn N std soln continuously thru system 
until no drift exists (usually ^10 min) and adjust "std. cal." 
control to read 85% full scale. Recorder tracings must be stable 
and show <0.3 division noise. If noisy conditions exist, replace 
dialyzer membrane. When recorder tracing indicates stable 
condition, immediately start sampling. 



7.030 



Shut-Down 



Place reagent lines in H 2 0, removing salicylate tine first. Let 
system wash out s=20 min. 

7.031 Co/ori metric Determination 

{Caution: See 51.065.) 

Weigh samples {See Table 7:04) into dry digestion tubes. Add 
9 g K 2 S0 4 , 0.42 g HgO, and 1 5 mL H 2 S0 4 to each tube. (Calibrated 
metal scoops may be used for solids.) Insert tubes into digestor 
block preheated to 410°, place shields around tubes, and digest 
45 min. 

After digestion, remove rack of tubes from block, place in 
hood, and let cool 8-10 min. (Time depends upon air flow 
around tubes.) Direct rapid spray of H 2 (kitchen sink dish 
rinsing sprayer works well) to bottom of each tube to dissolve 
acid digest completely. If ppt forms, place tube in ultrasonic 
bath to aid in redissolving salt. Let cool, dil. to vol., and mix 
thoroly. Transfer portion of each sample soln to AutoAnalyzer 
beaker. 



10 T 



u 



ype C Membrane J^LL 

****** 



6" DIALYZER 



ml/rr 



To Sampler 1-00 H 2 Q 

0-32 A| r 



n 



22 Acid/NaCI 



0.16 Sample 



_SU2 ^ir 

1 0,80 Acid/NaCI 



r 16 Resale 



0.32 Ail 



0.80 Acid/NaCI 



0,42 Buffer 



0.32 Salicylate 



0.16 Hypochlorite 




1 20 From F/C 



37° C 

"G"Coil 5T 



□ 



COLORIMETER 

660 nm 
15 mm F/C 




40/H, 2/1 S/w 



Modified 

^ff Waste 



FIG. 7:01 — Flow diagram for semi automated analysis for crude protein 



AOAC Methods (1980) 



Nitrogen 



129 



Table 7:04 Sample Weight 



Protein, % 


Sample, g 


6-24 


1.5±0.1 


25-40 


1.0±0.1 


41-50 


0.8±0.1 


51-60 


0.7±0.1 


61-90 


0.5±0.01 


>90 


Weigh sample equiv. 




to 50 mg N 



Place stds in tray in increasing order of concn, followed by 
group of samples. Analyze lowest concn std in duplicate, dis- 
carding first peak. Precede and follow each group of samples 
with std ref. curve to correct for possible drift. Analyze stds and 
samples at rate of 40/hr, 2/1 sample-to-wash ratio. Prep, std 
curve by averaging peak hts of first and second set of stds. Plot 
av. peak ht stds against N concn contained in each 250 mLtube. 
% Protein = [(mg N/250 mL from graph) 
x 6.25 x 100]/mg sample 



7.032 



Titrimetric Determination 



Digest as in 7.031. Cool 5 min and add only enough H 2 to 
dissolve salts (70-75 mL). Cool and attach digestion tube to 
distn head according to manufacturer's directions. Place receiver 
flask contg 25 mL std acid, 2.055{j), and 5-7 drops Me red 
indicator on platform. Condenser tip must be below surface of 
std acid soln. Add 50 mL NaOH-K 2 S soln to tube and steam 
distil vigorously until 125 mL distillate collects. Titr. excess acid 
with std 0.1/V NaOH soln, 2.055(k). Correct for reagent blank. 

% N - [(mL std acid x normality acid) - (mL std NaOH 
x normality NaOH)] x 1.4007/g sample 

% crude protein = % N x 6.25 

Urea and Ammoniacal Nitrogen [13) 
Official Final Action 

7.033 Reagents 

(a) De foaming soln. — Dow Corning Corp. Antifoam B Emul- 
sion. 

(b) Urease soln. — Prep, fresh soln by dissolving stdzd urease 
in H 2 so that each 10 mL neutzd soln will convert N of ^0.1 g 
pure urea. 

Standardization. — To det. alky of com. urease prepn dissolve 
0.1 g in 50 mL H 2 and titr. with 0.1/V HCt, using Me red, 2.055(i). 
Add same vol. 0.1/V HCI to each 0.1 g urease in prepg urease 
soln. To det. enzyme activity, prep, ca 50 mL neutzd 1% soln. 
Add different amts of soln to 0.1 g samples pure urea and follow 
with enzyme digestion and distn as in detn. Calc. activity of 
urease prepn from amt of this urease soln that completely 
converted urea, as detd by complete recovery of N by distn. 

(c) Calcium chloride soln. — Dissolve 25 g CaCI 2 in 100 mL 
H 2 0. 



7.034 



Determination 



Place 2 g sample in Kjeldahl flask with ca 250 mL H 2 0. Add 
10 mL urease soln, stopper tightly, and let stand 1 hr at room 
temp, or 20 min at 40°. Cool to room temp, if necessary. Use 
addnl urease soln if feed contains >5% urea (ca 12% protein 
equiv.). Rinse stopper and neck with few mL H 2 0. Add ^2 g 
MgO (heavy type), 5 mL CaCI 2 soln, and 3 mL defoaming soln, 
and connect flask with condenser by Kjeldahl connecting bulb. 
Distil 100 mL into measured vol. std acid, 2.055{j), and titr. with 
std alkali, 2.055(k), using Me red, 2.055(i). 

* Surplus method^see inside front cover. 



Urea ( 14)— Official Final Action 

(Applicable to animal feeds and their ingredients) 
7.035 Apparatus 

Spectrophotometer.— Instrument with max. band width 2.4 
nm at 420 nm, with 1 cm cells. 



7.036 



Reagents 



(a) p-Dimethylaminobenzaldehyde [DMAB) soln. — Dissolve 
16.00 g (Eastman Kodak Co. No. 95 only) in 1 L alcohol and add 
100 mL HCI. Stable 1 month. Prep, new std curve with each new 
batch of reagent. 

(b) Zinc acetate soln.— Dissolve 22.0 g Zn(OAc) 2 .2H 2 in H 2 0, 
add 3 mL HOAc, and dil. to 100 mL. 

(c) Potassium ferrocyanide soln. — Dissolve 10.6 g K 4 Fe(CN) 6 
.3H 2 in H 2 and dil. to 100 mL. 

(d) Vegetable charcoal. — Darco G-60. 

(e) Phosphate buffer soln.— pH 7.0. Dissolve 3.403 g anhyd. 
KH 2 P0 4 and 4.355 g anhyd. K 2 HP0 4 sep. in ca 100 mL portions 
freshly distd H 2 0. Combine solns and dil. to 1 L with H 2 0. 

(f) Urea std solns.— U) Stock soln. — 5 mg/ml Dissolve 
5.000±0.001 g reagent grade urea in H 2 and dil. to 1 L with 
H 2 0. (2) Working solns.— 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 
and 2.0 mg urea/5 mL Pipet 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 
mL stock soln into 250 mL vol. flasks and dil. to vol. with 
phosphate buffer. {3) Reference soln.— Use std soln contg 1.0 
mg urea/5 mL as ref. std. Store at <24°. Stable 1 week. 

7.037 Preparation of Standard Curve 

Pipet 5 mL aliquots of working std solns into 20 x 150 mm 
(25 mL) test tubes and add 5 mL DMAB soln to each. Prep, 
reagent blank of 5 mL buffer soln and 5 mL DMAB soln. Shake 
tubes thoroly and let stand 10 min in H 2 bath at 25°. Read A 
in 1 cm cell at 420 nm with reagent blank at zero A. Plot A 
against concn urea. Plot should be straight line; if not, repeat, 
using new lot of DMAB. 



7.038 



Determination 



Weigh 1.00 g ground sample into 500 mL vol. flask. Add 1 g 
charcoal, ca 250 mL H 2 0, 5 mL Zn(OAc) 2 soln, and 5 mL K 4 Fe(CN) 6 
soln. Shake mech. 30 min and dil. to vol. with H 2 0. Let stand 
until ppt settles. Decant thru Whatman No. 40 paper and collect 
clear filtrate. Pipet 5 mL filtrate into test tube, add 5 mL DMAB 
soln, and shake thoroly. Include reference std (5 mL soln (f)(5) 
and 5 mL DMAB soln) and reagent blank with each group of 
samples. Let stand 10 min in H 2 bath at 25°. Read A at 420 nm 
against reagent blank. 

% Urea = (1.0 x y4 sample x 100)/(/4 std x mg sample in aliquot). 

7.039 if Albuminoid Nitrogen— Official Final Action * 

Pptn with Cu(OH) 2 and N detn. See 22.020-22.021, 10th ed. 

7.040 * Amido Nitrogen — Official Final Action * 

% total N - % albuminoid N = % amido N. 



Nitrate and Nitrite Nitrogen {IS) — Official Final Action 

7.041 Principle 

Nitrate and nitrite are extd with Cd and Ba chloride soln. Bulk 
of sol. proteins are pptd in alk. soln and clarified soln is passed 
thru metallic Cd column, reducing nitrate to nitrite. Nitrite is 
measured colon' metrically. {Caution: Cd salts are toxic. See 
51.084.) 



130 



7. Animal Feed 



AOAC Methods (1980) 



7.042 



Reagents and Apparatus 



(a) Nitrate nitrogen std soins. — (7) Stock so/n. — 12 fig nitrate 
N/mL Dissolve 0.867 g KN0 3 in 1 L H 2 0. Dil. 25 mL to 250 mL 
with H 2 0. (2) Working soins.— 0.6, 1.2, 1.8,2.4, 3.0 ^g N/mL. Dil. 
5, 10, 15, 20, and 25 mL stock soln to 100 mL with H 2 0. 

(b) Extracting soin. — Dissolve and dil. 50 g CdCI 2 and 50 g 
BaCI 2 to 1 L with H 2 0. Adjust to pH 1 with HCI. 

(c) Ammonium chloride buffer soin, — pH 9.6. Dissolve 50 g 
NH 4 CI in 500 mL H 2 and adjust pH with NH 4 OH. Dil. to 1 L with 
H 2 0. 

(d) Sodium hydroxide so/a? —2.5/V. Dissolve 50 g NaOH in 500 
mL H 2 0. 

(e) Suifaniiamide soin. — 0.5%. Dissolve 1.25 g sulfanilamide 
in 250 mL HCI (1 + 1). Soln is stable 1-2 months. 

(f) Coupling reagent. — Dissolve 0.5 g N(1-naphthyl)ethylene- 
diamine.HCI in 100 mL H 2 0. Store in g-s dark bottle in refrigerator. 
Soln is stable several weeks. 

(g) Salt soin .-—Dissolve 100 g NaCI in 500 mL H 2 0. Add 50 mL 
buffer soln, (c), and dil. to 1 L with H 2 0. 

(h) Reduction tube. — 25 mL buret or equiv. id chromatgc tube 
with stopcock and reservoir (Kontes Glass Co. Cat. No. K-420280 
or SGA Scientific Inc. Cat. No. JC-1506). 



7.043 



Preparation of Columns 



Prep, supply of metallic Cd by placing Zn rods into 500 mL 
20% CdS0 4 soln. After reaction for 3 hr, discard soln and scrape 
moss-like Cd growth from Zn rods. Place Cd in high-speed 
blender, add 500 mL H 2 0, and blend 2 sec. Wash fine metal 
particles with H 2 onto sieves, collecting only 20-40 mesh size. 
Fill reduction tube with H 2 and add 2 cm plug of glass wool. 
Press any trapped air from glass wool as it is pushed to bottom 
of column with glass rod. Add Cd to depth of 10 cm, using min. 
of very gentle tapping. Wash column with 25 mL 0.10/V HCI, two 
25 mL portions H 2 0, and finally 25 mL buffer, (c), dild 1 + 9. 
Keep column covered with salt soln, (g), when not in use. 

Normally columns can be used repeatedly if kept under salt 
soln between analyses. When succession of highly proteina- 
ceous or other sol. org. contg samples are treated, flow rate 
may decrease gradually. Repeating 25 mL 0.10/V HCI treatment 
may restore original flow rate; if not, prep, new column. 
Reproducible flow rate is important. Actual rate can be 3-5 
mL/min but once established, it must be identical (±0.1 mL) for 
samples and stds. 

7.044 Preparation of Standard Curve 

Prep, std curve of 3, 6, 9, 12, and 15 fig nitrate-nitrite N by 
pipetting 5.0 mL aliquots of working std solns into 30 mL 
beakers. Add 5 mL buffer soln, (c), and 15 mL H 2 0, mix well, 
and transfer quant, to reduction column, using min. H 2 0. Adjust 
flow rate thru column to 3-5 mL/min. Just as reservoir empties, 
add 15 mL salt soln, (g). Collect eluate, including salt wash, in 
50 mL vol. flask (total vol. of eluate should be ca 40 mL). Add 
5 mL sulfanilamide soln, (e), mix, and let stand 3 min. Add 2 mL 
coupling reagent, (f), mix, dil. to vol. with H 2 0, mix, and let stand 
20 min for max. color development. Color is stable 3=2 hr. Det. 
A in 1 cm cells at 540 nm against reagent blank. Plot/4 against 
fig nitrate-nitrite N. 



7.045 



Extraction 



(a) Low level nitrate samples (grains, meals, supplements, 
etc.). — Wash 5.0 g finely ground sample into 250 mL vol. flask. 
Add 100 mL extg soln, (b), and 100 mL H 2 0, and mix. Let stand 
1 hr with occasional swirling. Add 20 mL 2.5/V NaOH, dil. to vol. 
with H 2 0, mix, and filter immediately thru rapid paper. Pipet 10 



mL buffer soln, (c), into 100 mL vol. flask, dil. to vol. with clear 
filtrate, and mix. 

(b) Dry, high level nitrate products {dried plants, hays, meals, 
etc.). — Weigh 5.0 g finely ground sample into 500 mL vol. flask. 
Add 100 mL extg soln, (b), and 300 mL H 2 0, and mix. Let stand 
1 hr with occasional swirling, add 40 mL 2.5/V NaOH, dil. to vol. 
with H 2 0, mix, and filter immediately thru rapid paper. Pipet 10 
mL buffer soln^ (c), into 100 mL vol. flask, dil. to vol. with clear 
filtrate, and mix. 

(c) Grasses, silages, and other wet materials. — Weigh 100 g 
sample into 1 gal. capacity high-speed blender. Add 100 mL 
extg soln, (b), and 800 mL H 2 0, including vol. H 2 contributed 
by sample as detd in 7.003 or 7.005. Homogenize 1 min, pour 
into 2 L beaker, and let stand 1 hr. Add 100 mL buffer soln, (c) 
(total vol. 1 L), mix well, and filter thru Whatman No. 42 paper, 
collecting portion of clear filtrate. 



7.046 



Determination 



( a) Nitrate plus nitrite nitrogen .—Pi pet25mLbufferedsample 
exts, 7.045(a) or (b), or 5 mL ext, (c), into reduction column and 
treat as in 7.044, beginning, ''Adjust flow rate thru column 
. . ." Rinse column with 30 mL H 2 between samples to remove 
NaCI. Use portion of buffered sample exts with equiv. diln and 
pH as ref. soln in detg A at 540 nm. Also det. nitrate-nitrite in 
reagents and correct for this blank value. Calc. total nitrate- 
nitrite N from std curve. 

(b) Nitrite nitrogen. — Pipet aliquot clear sample filtrate (contg 
<15 fig nitrite) into 50 mL vol. flask and dil. with H 2 to ca 40 
mL. Mix well, add 5 mL sulfanilamide soln, (e), mix, and let 
stand 3 min. Add 2 mL coupling reagent, (f), and dil. to vol. with 
H 2 0. Mix well and let stand 20 min for max. color development. 
Measure >4 in 1 cm cells against sample ext with equiv. diln at 
540 nm. Correct for nitrite reagent blank. 

(c) Nitrate nitrogen. — Calc. by difference between (a) and (b) 
above. 



7.047 



Calculation 



ppm N0 2 and/or N0 3 -N = fig N0 3 -N found x diln factor/g 

sample. 
Diln factors for exts: 7.045(a), 11.1; (b), 22.2; (c), 200. 



Pepsin Digestibility of Animal Protein Feeds (16) 
Official Final Action 



7.048 



Principle 

Defatted sample is digested 16 hr with warm soln of pepsin 
under const agitation. Insol. residue is isolated by filtering, 
washed, dried, and weighed to det. % residue. Residue is 
examined microscopically and analyzed for protein. Filtration 
method is applicable to all animal proteins. Methods are not 
applicable to vegetable proteins or mixed feeds because of 
presence of complex carbohydrates and other compds not 
digested by pepsin. 

7.049 Apparatus 

(a) Agitator. — See Fig. 7:02. Continuous, slow speed (15 rpm), 
end-over-end type, to operate inside incubator at 45±2° and 
carry 8 oz screw-cap prescription bottles, or equiv. Agitator and 
bottles available from D. E. Sims, 716 Forrest Ave, Quincy, IL 
62301. Stirring or reciprocating (shaking) type agitator cannot 
be used because solid particles collect on sides of bottle and do 
not contact pepsin soln. If heat from agitator motor raises 
incubator temp, to >45°, mount motor outside incubator by 
drilling hole thru side of incubator and connecting motor to 



AOAC Methods (1980) 



Nitrogen 



131 







FIG. 7:02^Agitator 



agitator with extension shaft and coupling (available from 
agitator supplier). {Caution: See 51.012.) 

(b) Settling rack. — Wood or metal to hold digestion bottles at 
45° angle. May be made from 2 boards nailed horizontally into 
"V" cut into vertical end pieces. Also available from agitator 
supplier, (a). 

(c) FUtering device. — Modified California buchner, 7.063(d), 
available from Labconco Corp., 8811 Prospect Ave, Kansas City, 
MO 64132, No. 55100. (If edge of screen is rough, smooth with 
small-tip soldering iron.) Use with retainer sleeve, 2 x 2.75" od 
stainless steel tube, available from agitator supplier, (a). 

(d) Glass fiber filter. — 7 cm, Reeve Angel No. 934- AH, or 
equiv. 

(e) Moisture dishes. — Al, 78 mm od x 20 mm, with outside 
cover and vertical sides (Curtin Matheson Scientific, Inc., No. 
19370-30, or equiv.). 

7.050 Reagent 

Pepsin soln.— 0.2% pepsin (activity 1:10,000) in 0.075/V HCI; 
do not use pepsin of activity other than 1:10,000. Prep. Just 
before use by dilg 6.1 ml_ HCI to 1 L and heating to 42-45°. Add 
pepsin and stir gently until dissolved. Do not heat pepsin soln 
on hot plate or overheat. 

7.051 Preparation of Sample 

Sieve sample, 7.001, thru No. 20 sieve. Grind portion retained 
on sieve to pass No. 20 sieve. Combine both portions and blend 
by stirring and shaking in pt (500 mL) jar. Thoro blending is 
essential. Because of high fat content of many animal products, 
grinding without sieving may cause sticking in mill, loss of 
moisture or fat, or poorly blended sample. 



7.052 



Extraction 



{Caution; See 51.011, 51.039, and 51.054.) 

Prep, extn thimble from 11 cm Whatman No. 2 paper, or 
equiv., as follows: Fold paper in half; straighten paper and 
refold at right angles to first fold; turn paper over and repeat 
process with folds at 45° to original fold; while holding creased 
paper in one hand, place short test tube (6-8 mm smaller in 
diam. than extractor sample holder or cup in which thimble is 
to be used) at its center; fold along natural crease lines to form 



4-pointed star around tube; and wrap points in same direction 
around tube to complete thimble. 

Weigh 1.000 g ground sample (0.500 g of poultry byproducts 
or hydrolyzed feathers because of gummy nature and amt of 
residue) into thimble and ext 1 hr with ether at condensation 
rate of 3-4 drops/sec. (If Soxhlet is used, top of thimble should 
extend above siphon tube to avoid loss of solid particles. If 
paper contg sample is totally submerged in siphon cup, sample 
must be completely wrapped in paper.) Observe ether ext to 
det. that no solid particles were carried into solv. For approx. fat 
content detn, evap. ether, and dry and weigh residue. Remove 
paper from sample container or cup and let dry at room temp. 
Unfold, and quant, brush defatted sample into digestion bottle, 
avoiding contamination by brush bristles or filter paper fibers. 
Use of powder funnel is helpful to avoid loss. 



7.053 



Pepsin Digestion 



To defatted sample in agitator bottle add 1 50 mL freshly prepd 
pepsin soln prewarmed to 42-45°. Be sure sample is completely 
wetted by pepsin soln. Stopper bottle, clamp in agitator, and 
incubate with const agitation 16 hr at 45°. 



7.054 



Treatment of Residue 



Dry individual sheets of glass fiber filter, (d), 30 min at 110° 
in moisture dishes with cover open. Cool in desiccator 30 min 
with cover closed, and weigh (lAA,). 

Remove bottles from agitator. Place in 45° angle settling rack 
and loosen caps. Let residue settle ^15 min. Place weighed filter 
in California buchner, (c), apply suction, and moisten with H 2 0. 
Place retainer sleeve on filter and press down gently. Rinse 
particles of residue on cap onto filter with small amt H 2 0. Carry 
bottle from rack to filter at same angle as settled and slowly 
pour contents thru filter as continuous small stream, avoiding 
all unnecessary agitation. Liq. passes thru paper as rapidly as 
poured, with residue spreading over surface of filter but not 
covering it completely until all or pratically all of liq. has passed 
thru. If filtration rate becomes slow, it may be accelerated by 
adding acetone washes described below, but only if no signif- 
icant amt of digestion mixt. remains on funnel when acetone is 
added. (Filtration (passage of aq. mixt. thru filter) should be 
complete within 1 min with most proteins.) After supernate has 
passed thru filter, quant, transfer residue onto filter as follows: 

Add 15 mL acetone to bottle. Hold thumb over bottle neck 
and shake vigorously. Release pressure, replace thumb over 
bottle neck, and shake bottle in inverted position over filter. 
Remove thumb, letting acetone and residue discharge onto 
filter. Repeat rinse with second 15 mL portion acetone, shaking 
and releasing pressure as above. Inspect bottle, and rinse further 
with acetone, using policeman, if necessary. If >3 mm liq. 
remains on paper when acetone washes are started, it may be 
necessary to use three 15 mL acetone washes instead of 2 to 
increase filtration rate. 

After all liq. passes thru funnel, wash residue and inside 
surface of retainer sleeve with 2 small portions acetone from 
wash bottle or hypodermic syringe, and suck dry. Remove 
retainer sleeve from funnel. Transfer filter to original moisture 
dish. Scrape or brush any residue particles or filter clinging to 
retainer sleeve or funnel onto filter in moisture dish. Dry in oven, 
cool, and weigh as before (W 2 ). Calc. % indigestible residue = 
(W 2 - WJ x 100/g sample. 

Det. indigestible protein by transferring filter contg residue 
directly to Kjeldahl flask. Proceed as in 7.015. {Caution: Violent 
reaction may take place when NaOH is mixed with dild digestion 
mixt., caused by large excess H 2 S0 4 due to small amt org. 
material from residue and none from glass filter. Avoid by 



132 



7. Animal Feed 



AOAC Methods (1980) 



thoroly mixing and cooling digestion mixt. before addn of NaOH 
or by using 20 mL H 2 S0 4 in Kjeldahl digestion instead of 25 ml_.) 
Make blank detn on 1 sheet of glass filter and subtract from 
each sample detn, if necessary. Calc. % protein based on original 
sample wt. Result represents % indigestible protein in sample. 
Convert to % crude protein content of sample not digested, 
"protein indigestible" - % indigestible protein in sample x 
100/% total crude protein in sample. 

OTHER CONSTITUENTS 
Crude Fat or Ether Extract 

Use method 7.056 or 7.057 for mixed feeds other than (1) entirely 

baked and/or expanded, (2) entirely dried milk products, or (3) 

contg urea. 



Direct Method — Official Final Action 



7.055 



Reagent 

Anhydrous ether. — Wash com. ether with 2 or 3 portions H 2 0, 
add solid NaOH or KOH, and let stand until most of H 2 is 
abstracted from the ether. Decant into dry bottle, add small 
pieces of carefully cleaned metallic Na, and let stand until H 
evolution ceases. Keep ether, thus dehydrated, over metallic Na 
in loosely stoppered bottles. (Caution: See 51.034 and 51.054.) 



beads. Rinse lip of tube with few mL pet ether. Re-ext liq. 
remaining in tube twice, each time with only 15 mL of each 
ether, shaking 1 min after addn of each ether. Pour clear ether 
soln thru filter into same beaker as before, and wash tip of tube, 
stopper, funnel, and end of funnel stem with few mL of mixt. of 
2 ethers (1+1). Evap. slowly on steam bath under gentle stream 
of air or N. Continue heating on steam bath 15 min after solv. 
has evapd; then cool to room temp. 

Redissolve dried fat residue in four 10 mL portions Et ether, 
filtering each portion thru small pledget of cotton into 100 mL 
beaker, contg few glass beads, that has been predried 30 min 
at 100°, cooled to room temp, in desiccator, and weighed 
immediately. Use fifth 10 mL portion ether for rinsing cotton 
and funnel. Evap. ether on steam bath, dry 90 min at 100°, cool 
to room temp, in desiccator, and weigh immediately. Correct 
this wt by blank detn on reagents used. 

7.059 In Dried Milk Products {18)— Official Final Action 

Proceed as in 16.199(b) and 16.200, using 8.5 mL H 2 and 1.5 
mL NH 4 OH. 

7.060 In Fish Meal 

See 18.046 and 18.047-18.049. 



7.056 



Determination 



(Large amts H 2 0-sol. components such as carbohydrates, urea, 
lactic acid, glycerol, and others may interfere with extn of fat; 
if present, ext 2 g sample on small paper in funnel with five 20 
mL portions H 2 prior to drying for ether extn. Caution: See 
51.009, 51.011, and 51.054.) 

Ext ca 2 g sample, dried as in 7.003 or 7.006*, with anhyd. 
ether. Use thimble with porosity permitting rapid passage of 
ether. Extn period may vary from 4 hr at condensation rate of 
5-6 drops/sec to 16 hr at 2-3 drops/sec. Dry ext 30 min at 100°, 
cool, and weigh. 

7.057 Indirect Method— Official Final Action 

Det. moisture as in 7.003 or 7.006*; then ext dried substance 
as in 7.056, and dry again. Report loss in wt as ether ext. 

7.058 In Baked or Expanded and Intermediate Moisture 
[Semimoist or Soft-Moist) Pet Foods [17] 
Official Final Action 

(To be used only on products which have been baked and/or 
expanded, and on intermediate moisture pet foods. Not appli- 
cable to canned, fresh, or frozen pet food. Such products should 
be dried at 70-110°, then ground, and drying completed by 
7.003 or 7.006* followed by 7.056 or 7.057. Caution: See 51.011, 
51.054, and 51.073.) 

Place ca 2 g, accurately weighed, ground, well mixed sample 
in Mojonnier fat-extn tube, add 2 mL alcohol to prevent lumping 
on addn of acid, and shake to moisten all particles. Add 10 mL 
HCI (25-1-11), mix well, and set tube 30-40 min in H 2 bath at 
70-80°, shaking frequently. Cool to room temp, and add alcohol 
until liq. level rises into constricted portion of Mojonnier tube. 

Add 25 mL ether, stopper with glass, Neoprene, or good 
quality rubber stopper thoroly cleaned with alcohol, and shake 
vigorously 1 min. Carefully release pressure so that no solv. is 
lost. Wash adhering solv. and fat from stopper back into extn 
tube with few mL redistd pet ether (bp <60°). Add 25 mL redistd 
pet ether, stopper, and shake vigorously 1 min. Let stand until 
upper liq. is practically clear or centrf. 20 min at ca 600 rpm. 
Pour as much of ether-fat soln as possible thru filter consisting 
of cotton pledget packed just firmly enough in funnel stem to 
let ether pass freely into 150 mL beaker contg several glass 



Crude Fiber (79) — Official Final Action 
AOCS-AOAC Method 

(Caution: See 51.086.) 

7.061 Principle 

Crude fiber is loss on ignition of dried residue remaining after 
digestion of sample with 1.25% H 2 S0 4 and 1.25% NaOH solns 
under specific conditions. Method is applicable to grains, meals, 
flours, feeds, and fiber-bearing material from which fat can be 
extd to leave workable residue. 



7.062 



Reagents 



(a) Sulfuric acid soln. — 0.255 ±0.005/V. 1.25 g H 2 SO 4 /100 mL. 
Concn must be checked by titrn. 

(b) Sodium hydroxide soln.— O.313±0.005/V. 1.25g NaOH/100 
mL, free, or nearly so, from Na 2 C0 3 . Concn must be checked by 
titrn. 

(c) Prepared asbestos. — Spread thin layer acid- washed, me- 
dium or long fiber asbestos in evapg dish and heat 16 hr at 600° 
in furnace. Boil 30 min with 1.25% H 2 S0 4 , filter, wash thoroly 
with H 2 0, and boil 30 min with 1.25% NaOH. Filter, wash once 
with 1.25% H 2 S0 4 , wash thoroly with H 2 0, dry, and ignite 2 hr 
at 600°. 

Det. blank by treating 1.0 g prepd asbestos with acid and alkali 
as in detn. Correct crude fiber results for any blank, which 
should be negligible (ca 1 mg). Asbestos recovered from detn 
may be used in subsequent detns. 

(d) Alcohol. — 95% or reagent alcohol, MeOH, or isopropanol. 

(e) Ant/foam. — Dow Corning Corp. Antifoam A compd dild 
1+4 with mineral spirits or pet ether, or H 2 0-dild Antifoam B 
Emulsion (1+4). Do not use Antifoam Spray. 

(f) Bumping chips or granules. — Broken Alundum crucibles 
or equiv. granules (RR Alundum 90 mesh, Norton Co., 1 New 
Bond St, Worcester, MA 01606) are satisfactory. 



7.063 



Apparatus 



(a) Digestion apparatus. — With condenser to fit 600 mL 
beaker, and hot plate adjustable to temp, that will bring 200 mL 



AOAC Methods (1980) 



Fiber 



133 




200 mesh st, steel screen 

20 go. st. steel back plate" 
with 5/64" holes 




FIG. 7:03 — Oklahoma State filter screen 

H 2 at 25° to rolling boil in 15±2 min. (Available from Labconco 
Corp., 8811 Prospect Ave, Kansas City, MO 64132.) 

(b) Ashing dishes. — Silica, Vitreosil 70 x 15 mm; or porcelain, 
Coors, No. 450, size 1, or equiv. 

(c) Desiccator. — With efficient desiccant such as 4-8 mesh 
Drierite (CaCI 2 is not satisfactory). 

(d) Filtering device.— With No. 200 type 304 or 316 stainless 
steel screen (W. S. Tyler Co., 8200 Tyler Blvd, Mentor, OH 
44060), easily washed free of digested residue. Either Oklahoma 
State filter screen {see Fig. 7:03; available from Labconco Corp.) 
or modified California plastic buchner {see Fig. 7:04; consists 
of 2 piece polypropylene plastic funnel manufactured by Nalge 
Co., 75 Panorama Creek Drive, Rochester, NY 14602, Cat. No. 
4280, 70 mm (without No. 200 screen), or equiv. (also available 
from Labconco Corp.). Seal screen to filtering surface of funnel, 
using small-tip soldering iron). 

(e) Suction filter. — To accommodate filtering devices. Attach 
suction flask to trap in line with aspirator or other source of vac. 
with valve to break vac. 

(f) Liquid preheater. — For preheating H 2 0, 1.25% H 2 S0 4 , and 
1.25% NaOH solns to bp of H 2 0. Convenient system, shown in 
Fig. 7:05, consists of sheet Cu tank with 3 coils of %" (10 mm) 
od Cu tubing, 12.5' (3.8 m) long. Solder inlets and outlets where 
tubing passes thru tank walls. Connect to reflux condenser and 
fill with H 2 0. Keep H 2 boiling with two 750 watt thermostatically 
controlled hot plates. Use Tygon for inlet leads to reservoirs of 
H 2 0, acid, and alkali; use gum rubbertubing for outlets. Capacity 
of preheater is adequate for 60 analyses in 8 hr. 



7.064 



Preparation of Sample 



Reduce sample (riffle is suitable) to 100 g and place portion 
in sealed container for H 2 detn. Immediately det. H 2 0. Grind 
remainder to uniform fineness. (Weber mill (Sargent-Welch 
Scientific Co. S-60870) with screen 0.033-0.040" (No. 18 or 20), 
Micro mill (Pulverizing Machinery, Div. Mikropul Corp., 10 
Chatham Rd, Summit, NJ 07901) with screen V25-V16" (No. 
18-No. 12), and Wiley mill with 1 mm (No. 18) screen give 
comparable fineness.) Since most materials lose moisture during 
grinding, det. H 2 on ground sample at same time sample is 
taken for crude fiber detn. 




FIG. 7:04 — Modified California State buchner funnel, 2-piece polypro- 
pylene plastic, covered with 200-mesh screen. A, heat-sealed to edge of 
filtering surface 



7.065 



Determination 



Ext 2 g ground material with ether or pet ether, 14.088. If fat 
is <1%, extn may be omitted. Transfer to 600 mL beaker, 
avoiding fiber contamination from paper or brush. Add ca 1 g 
prepd asbestos, 200 mL boiling 1.25% H 2 S0 4 , and 1 drop dild 
antifoam. (Excess antifoam may give high results; use only if 
necessary to control foaming.) Bumping chips or granules may 
also be added. Place beaker on digestion app. with preadjusted 
hot plate and boil exactly 30 min, rotating beaker periodically 
to keep solids from adhering to sides. Remove beaker, and filter 
as in (a) or (b). 

(a) Using Oklahoma filter screen. — Turn on suction and insert 
screen (precoated with asbestos if extremely fine materials are 
analyzed) into beaker, keeping face of screen just under surface 
of liq. until all liq. is removed. Without breaking suction or 
raising filter, add 50-75 mL boiling H 2 0. After wash is removed, 




FIG. 7:0&— Continuous heater for distilled water, 1.25% alkali, and 1.25% 

acid 



134 



7. Animal Feed 



AOAC Methods (1980) 



repeat with three 50 ml_ washings. (Work rapidly to keep mat 
from becoming dry.) Remove filter from beaker and drain all 
H 2 from line by raising above trap level. Return mat and 
residue to beaker by breaking suction and blowing back. Add 
200 ml_ boiling 1.25% NaOH and boil exactly 30 min. Remove 
beaker, and filter as above. Without breaking suction, wash with 
25 mL boiling 1.25% H 2 S0 4 and three 50 mL portions boiling 
H 2 0. Drain free of excess H 2 by raising filter. Lower filter into 
beaker and wash with 25 mL alcohol. Drain line, break suction, 
and remove mat by blowing back thru filter screen into ashing 
dish. Proceed as in (c). 

(b) Using California buchner. — Filter contents of beaker thru 
buchner (precoated with asbestos if extremely fine materials 
are being analyzed), rinse beaker with 50-75 mL boiling H 2 0, 
and wash thru buchner. Repeat with three 50 mL portions H 2 0, 
and suck dry. Remove mat and residue by snapping bottom of 
buchner against top while covering stem with thumb or forefin- 
ger and replace in beaker. Add 200 mL boiling 1.25% NaOH and 
boil exactly 30 min. Remove beaker, and filter as above. Wash 
with 25 mL boiling 1.25% H 2 S0 4 , three 50 mL portions H 2 0, and 
25 mL alcohol. Remove mat and residue; transfer to ashing dish. 

(c) Treatment of residue. — Dry mat and residue 2 hr at 130 ±2°. 
Cool in desiccator and weigh. Ignite 30 min at 600+15°. Cool in 
desiccator and reweigh. 

% Crude fiber in ground sample = C = (Loss in wt on ignition 
- loss in wt of asbestos blank) x 100/wt sample. 

% Crude fiber on desired moisture basis = C x (100 - % 
moisture desired)/(100 - % moisture in ground sample). 

Report results to 0.1%. 



Asbestos-Free {AF) Method (20) 
Official Final Action 

7.066 Principle 

Principle is same as in 7.061, except sample is exposed to 
min. vac. needed to regulate filtration, and heating of sample 
solns prevents gelling or pptn of possible satd solns. 

7.067 Apparatus and Reagents 

See reagents 7.062(a), (b), and (f); app. 7.063(a), (c), (d), and 
(f), and 14.088; and in addn: 

(a) Filtration apparatus. —System to permit application of min. 
vac. necessary for filtration and washing of each sample within 
3-5 min. Each unit consists of reservoir manifold connected to 
(7) H 2 aspirator thru 120° stopcock, (2) atm. thru second 
stopcock with metering device, and {3) receptacle contg cone- 
shaped hard rubber gasket which provides vac. seal with cru- 
cible. Vac. gage attached to manifold indicates vac. applied to 
crucible. Crucible can be heated before and during filtration by 
flow of hot H 2 in surrounding jacket. (For photograph of app., 
see JAOAC 56, 1353(1973). Filtration unit is available as Model 
150 from Analytical Biochemistry Laboratories, Inc., PO Box 
1097, Columbia, MO 65201.) 

(b) Crucible. — Fritted glass, 50 mL, coarse porosity. Clean as 
follows: Brush, and flow hot tap H 2 into crucible to remove as 
much ash as possible. Submerge crucible in base soln, (c)(2), 
^5 min, remove, and rinse with hot tap H 2 0. Submerge in HCI 
(1 + 1), (c) (7), ^5 min, remove, and rinse thoroiy with hot tap 
H 2 followed by distd H 2 0. After 3-4 uses, back wash by 
inverting crucible on hard rubber gasket in filtration app., and 
flowing near-boiling H 2 thru crucible under partial vac. 

(c) Cleaning solns. — ( 7 )Acid soln. —HCI ( 1 + 1 ). (2) Base soln. — 
Dissolve 5 g Na 2 H 2 EDTA, 50 g Na 2 HP0 4 (tech. grade), and 200 
g KOH in H 2 0, and dil. to 1 L. Storage in sep. wide mouth 



containers holding 2-3 L soln into which crucibles can be placed 
is convenient. 



7.068 



Determination 



{Caution: See 51.011 and 51.073.) 

Ext 2 g ground material with ether or pet ether, 14.088. If fat 
is <1%, extn may be omitted. Transfer to 600 mL reflux beaker, 
avoiding fiber contamination from paper or brush. Add 0.25-0.5 
g bumping granules, followed by 200 mL near-boiling 1.25% 
H 2 S0 4 soln in small stream directly on sample to aid in complete 
wetting of sample. Place beakers on digestion app. at 5 min 
intervals and boil exactly 30 min, rotating beakers periodically 
to keep solids from adhering to sides. Near end of refluxing 
place California buchner, 7.063(d), previously fitted with No. 9 
rubber stopper to provide vac. seal, into filtration app., and 
adjust vac. to ca 25 mm Hg (735 mm pressure). At end of 
refluxing, flow near-boiling H 2 thru funnel to warm it; then 
decant liq. thru funnel, washing solids into funnel with min. of 
near-boiling H 2 0. Filter to dryness, using 25 mm vac, and wash 
residue with four 40-50 mL portions near-boiling H 2 0, filtering 
after each washing. Do not add wash to funnel under vac; lift 
funnel from app. when adding wash. 

Wash residue from funnel into reflux beaker with near-boiling 
1.25% NaOH soln. Place beakers on reflux app. at 5 min intervals 
and reflux 30 min. Near end of refluxing, turn on filtration app., 
place crucible, (b), in app., and adjust vac. to ca 25 mm. Flow 
near-boiling H 2 thru crucible to warm it. (Keep near-boiling 
H 2 flowing thru jacket during filtration and washing.) At end 
of refluxing, decant liq. thru crucible and wash solids into 
crucible with min. of near-boiling H 2 0. Increase vac. as needed 
to maintain filtration rate. Wash residue once with 25-30 mL 
near-boiling 1.25% H 2 S0 4 soln, and then with two 25-30 mL 
portions near-boiling H 2 0, filtering after each washing. (Filtering 
and washing takes ca 3-5 min/sample.) Do not add wash to 
crucible under vac 

Dry crucible with residue 2 hr at 130±2° or overnight at 110°, 
cool in desiccator, and weigh. Ash 2 hr at 550±10°, cool in 
desiccator, and weigh. Do not remove crucibles from furnace 
until temp, is =£250°, as fritted disk may be damaged if cooled 
too rapidly. 

% Crude fiber = Loss in wt on ignition x 100/wt sample. 

Acid-Detergent Fiber and Lignin [21) 
Official Final Action 

[Caution: See 51.086.) 
7.069 Reagents 

(a) Sulfuric acid. — 72% by wt. Stdze reagent grade H 2 S0 4 to 
sp gr 1.634 at 20° or 24.00/V: Add 1200 g H 2 S0 4 to 440 mL H 2 
in 1 L MCA vol. flask with cooling. Stdze to 1634 g/L at 20° by 
removing soln and adding H 2 or H 2 S0 4 as required. {Caution: 
See 51.030.) 

(b) Acid-detergent soln . — Add 20 g cetyl trimethylammonium 
bromide (tech. grade) to 1 L 1.00/V H 2 S0 4 , previously stdzd. 
Agitate to aid soln. 

(c) Asbestos. — Place 100 g asbestos in 3 L flask contg 850 mL 
H 2 0. Add 1.4 L H 2 S0 4 (tech. grade), mix, and let cool 2 hr at 
room temp. Filter on large buchner and wash with H 2 0. Resus- 
pend mat in H 2 and pour into bag sewn from rectangle of 
fiberglass window screening, 14x18 mesh (bag should be ^45 
cm wide x 30 cm deep). Wash by immersion and agitation in 
partly filled sink to remove fine particles. Ash recovered asbestos 
16 hr in 800° furnace. Store in dry form until use. Used asbestos 
may be rewashed, reashed, and reused. Com. prepd acid-washed 
asbestos is unsatisfactory unless treated with 72% H 2 S0 4 and 
ashed at 800°. 



AOAC Methods (1980) 



Carbohydrates 



135 



7.070 Apparatus 

(a) Refluxing apparatus. — Any conventional a pp. suitable for 
crude fiber detns. Berzelius beakers (600 mL) and condensers 
made from 500 mL r-b flasks are also satisfactory. 

(b) Fritted glass crucibles. — Use coarse porosity, 40-50 mL 
Pyrex crucible. Wash new crucibles and ash at 500°. Remove 
while still hot and place in 100° forced-draft oven s*1 hr. Cool 
15 min in desiccator over P 2 5 or Mg(Cl0 4 ) 2 and weigh in same 
order samples are to be weighed. Check balance after each 
weighing if crucibles are still warm. Hold length of time from 
oven to balance pan as const as possible and always weigh 
crucibles in same order. 



7.071 



Determination of Acid-Detergent Fiber 



Weigh 1 g air-dried sample ground to pass 1 mm screen, or 
approx. equiv. amt wet material, into refluxing container. Add 
100 mL acid-detergent soln at room temp. 

Heat to boiling in 5-10 min; reduce heat to avoid foaming as 
boiling begins. Reflux 60 min from onset of boiling, adjusting 
boiling to slow, even level. Remove container, swirl, and filter 
thru weighed {WJ fritted glass crucible, using min. suction. 
Increase vac. only as needed. Shut off vac. Break up filtered mat 
with rod and fill crucible 2 / 3 full with hot (90-100°) H 2 0. Stir and 
let soak 15-30 sec. Dry with vac. and repeat H 2 washing, 
rinsing sides of crucible. Wash twice similarly with acetone. 

Repeat acetone washings until no more color is removed, 
breaking up all lumps so that solv. wets all particles of fiber. 
Remove residual acetone with vac. Dry 3 hr or overnight in 100° 
forced-draft oven and weigh {W 2 ). Calc. % acid-detergent fiber 
= 100 (W 2 - W,)/S, where S = g sample x g oven-dried 
matter/g air-dried or wet matter, detd on sep. sample. 



7.072 



Determination of Lignin 



To crucible contg fiber, 7.071, add 1 g asbestos. Place crucible 
in 50 mL beaker for support or arrange crucibles in shallow 
enamel pan. Cover contents of crucible with cooled (15°) 72% 
H z S0 4 and stir with glass rod to smooth paste, breaking all 
lumps. Fill crucible about half-way with acid and stir. Leave 
glass rod in crucible; refill with 72% H 2 S0 4 and stir hourly as 
acid drains, keeping crucible at 20-23° (cool if necessary). After 
3 hr, filter as completely as possible with vac, and wash with 
hot H 2 until acid-free to pH paper. Rinse sides of crucible and 
remove stirring rod. Dry crucible tn 100° forced-draft oven, cool 
in desiccator over P 2 5 or Mg(CI0 4 ) 2 , and weigh (W 3 ). Ignite 
crucible in 500° furnace 2 hr or until C-free. Place crucible while 
still hot into 100° forced-draft oven 1 hr. Transfer to desiccator, 
cool, and weigh (W 4 ). 

Det. asbestos blank by weighing 1 g asbestos into tared 
crucible. Proceed as above, beginning "Cover contents of cru- 
cible . . ." Record any loss in wt on ashing {W 5 ). Discontinue 
detn of blank if asbestos blank is <0.0020 g/g asbestos. Calc. 
% acid-insol. lignin = (VV 3 - W 4 - W 5 )/S. 



Total Sugars (22)— Official Final Action 



7.073 



Reagents 

(a) Soxhlet modification ofFehling soln.— Prep, as in 31 .034(a) 
and (b). 

(b) Invert sugar std soln. — 1.0%. Prep, as in 31.034(c), but do 
not neutze. Dil. to 0.5% just before use for analysis of most 
products. 

(c) Lactose std soln. — 1.0%. Dissolve 5.000 g lactose in H 2 
and dil. to 500 mL. Prep, daily. 



7.074 Apparatus 

(a) Lamp.— Fluorescent desk lamp or 150 watt reflector spot 
lamp, to illuminate boiling soln. 

(b) Heater. — Glas-Col mantle, 250 mL, placed over mag. stir- 
rer. Adjust heat so that 50 mL H 2 contg stirring bar will boil in 
3 min. Mag. stirring hot plate is also satisfactory. 

7.075 Preparation of Sample and inversion 

(a) Feeds containing molasses.— Weigh appropriate size sam- 
ple, prepd as in 7.002 but not ground, to provide final soln ca 
0.5% invert sugar but ^5 g, into 250 mL P flask (Corning Glass 
Works No. 5840, or equiv.). Add 150 mL H 2 0, swirl to wet and 
mix, and heat just to bp. Let stand to cool, dil. to vol., mix, and 
let stand to settle coarse particles. Transfer 50 mL supernate to 
100 mL vol. flask and add 2.5 mL HCI (sp gr 1.18 at 20/4°). Let 
stand overnight at 5=25°, dil. to vol., and mix. (If aliquot to be 
used in detn is >25 mL, it is necessary to neutze inverted soln.) 

(b) Feeds containing milk products. — Weigh appropriate size 
sample to provide final soln ca 1% lactose into 250 mL vol. 
flask. Thoroly moisten sample with H 2 0, swirl to dissolve lactose, 
dil. to vol., mix, and let stand to settle coarse particles. Proceed 
as in 7.077(b). 



7.076 



Standardization 



Fill 50 mL buret, with offset tip, with std sugar soln (invert 
sugar for use with 7.077(a) and lactose with 7.077(b)). Proceed 
as in 31.080, par. 2, except use same type flask as used in 7.077, 
do not add H 2 0, and start stirring after addn of indicator. 



7.077 



Determination 



(a) Difference method.— Add reagents and stirring bar to 250 
mL extn flask (Corning Glass Works No. 5160, or equiv.) or to 
erlenmeyer, as in 7.076. Transfer aliquot inverted soln, (a), to 
flask so that >1 but <5 mL std soln will be required to reach 
end point, place on preheated mantle or hot plate, heat to bp, 
boil 2 min, add ca 1 mL indicator, and begin stirring. Complete 
detn by titrg with std sugar soln to same end point used in 
stdzn. Color change is not so sharp as in stdzn, but under 
suitable light it is definite, discernible, and repeatable. 

(b) Alternative method. —Fill buret with sample soln, (b), or 
inverted sample soln, (a). As in 7.076, place reagents in flask, 
place on heater, add sample soln to within 2 mL of final titrn 
(detd by trial), bring to bp, boil 2 min, and complete titrn as in 
(a). 



7.078 



Caicuiations 



% Total sugar (as invert or lactose) 

=[(F - M) x / x 100]/[V x (W/250) x D)], 

where F - mL std sugar required to reduce mixed Soxhlet 
reagent in stdzn; M - mL std soln required to complete detn 
(omit in alternative method); / = concn std soln; V = mL sample 
soln in aliquot used; W = g sample; and D = diln factor. 
Report total sugars, expressed as invert or as lactose. 

7.079 Sucrose {23) — Official Final Action 

Place 10 g sample in 250 mL vol. flask. If material is acid, 
neutze by adding 1-3 g CaC0 3 . Add 125 mL 50% alcohol by vol., 
mix thoroly, and boil on steam bath or by partially immersing 
flask in H 2 bath 1 hr at 83-87°, using small funnel in neck of 
flask to condense vapor. Cool and let mixt. stand several hr, 
preferably overnight. Dil. to vol. with neut. 95% alcohol, mix 
thoroly, let settle or centrf. 15 min at 1500 rpm, and decant 
closely. Pipet 200 mL supernate into beaker and evap. on steam 



136 



7. Animal Feed 



AOAC Methods (1980) 



bath to 20-30 mL Do not evap. to dryness. Little alcohol in 
residue does no harm. 

Transfer to 100 mL vol. flask and rinse beaker thoroly with 
H 2 0, adding rinsings to flask. Add enough satd neut. Pb[OAc) z 
soln (ca 2 mL) to produce flocculent ppt, shake thoroly, and let 
stand 15 min. Dil. to vol. with H 2 0, mix thoroly, and filter thru 
dry paper. Add enough anhyd. Na 2 C0 3 or K oxalate to filtrate to 
ppt all Pb, again filter thru dry paper, and test filtrate with little 
anhyd. Na 2 C0 3 or K oxalate to make sure that all Pb has been 
removed. 

Place 50 mL prepd soln in 100 mL vol. flask, add piece of 
litmus paper, neutze with HCI, add 5 mL HCI, and let inversion 
proceed at room temp, as in 31.026(c). When inversion is 
complete, transfer soln to beaker, neutze with Na 2 C0 3 , return 
soln to 100 mL flask, dil. to vol. with H 2 0, filter if necessary, and 
det. reducing sugars in 50 mL soln (representing 2 g sample) as 
in 31.038. Calc. results as invert sugar. 

% Sucrose = [% total sugar after inversion - % reducing 
sugars before inversion (both calcd as invert sugar)] x 0.95. 

Because insol. material of grain or cattle food occupies some 
space in flask as originally made up, correct by multiplying all 
results by factor 0.97, as results of large number of detns on 
various materials show av. vol. of 10 g material to be 7.5 mL. 



•k Starch — Official Final Action * 

7.080 Direct Acid Hydrolysis 

See 8.019. Use sample contg 2.5-3 g dry material. 

7.081 Diastase Method with Subsequent 
Acid Hydrolysis 

See 7.067, 12th ed. 

7.082 Extraction with Subsequent Enzyme Hydrolysis 
See 14.075-14.080. 

7.083 in Presence of Interfering Polysaccharides {24) 
See 22.048, 10th ed. 



7.090 * Potassium iodide (25)— Official Final Action * 

Starch-iodine spot test. See 7.076, 12th ed. 

Minerals in Feeds by Atomic Absorption 
Spectrophotometry {29) — Official Final Action 

{Caution: See 51.006.) 

7.091 Apparatus 

Atomic absorption spectrophotometer.— See 2.109. 

7.092 Operating Parameters 

See Table 2:04, except use fuel-rich air-C 2 H 2 flame for Ca and 
Mg, and ranges of operation for fjug element/mL soln are: Ca 
5-20, Cu 2-20, Fe 5-20, Mg 0.5-2.5, Mn 5-20, and Zn 1-5. 

7.093 Reagents 

{See introduction to 2.110. Com. prepd std solns may be used.) 

(a) Calcium std solns.— Prep, as in 2.110(a). 

(b) Copper, iron, magnesium, manganese, and zinc std 
solns.— Prep, stock solns as in 2.110(b), (c), (e), (f), and (g), and 
dil. aliquots with 0.1-0.5/V HCI to make ^4 std solns of each 
element within range of detn. 

7.094 Preparation of Sample Solution 

(a) Dry ashing (not applicable to mineral-mix feeds).— Ash 
2-10 g sample in well-glazed porcelain dish. Start in cold furnace, 
bring to 550°, and hold 4 hr. Cool, add 10 mL 3/V HCI F cover with 
watch glass, and boil gently 10 min. Cool, filter into 100 mL vol. 
flask, and dil. to vol. with H 2 0. Subsequent dilns with 0.1-0.5/V 
HCI may be necessary to bring sample solns into anal, range, 
except for Ca. Final Ca diln must contain enough La soln, 
2.110(d), to provide 1% La concn after diln to vol. with H 2 0. 

(b) Wet digestion.— Proceed as in 7.097(a), adding 25 mL 
HN0 3 for each 2.5 g sample and dilg to 100 mL with H 2 0. 
Digestion can be made at low heat on hot plate, using 600 mL 
beaker covered with watch glass. Subsequent dilns with 0.1-0.5/V 
HCI may be necessary to bring sample solns into anal, range, 
as in (a). 



7.084 In Condensed or Dried Milk Products — 
Qualitative Test {25) 

See 22.049, 10th ed. 

7.085 * Pentosans (25)— Official Final Action * 
See? 22.050-22.051, 10th ed. 

7.086 * Galactan — Official Final Action * 

See 22.052, 10th ed. {Caution: See 51.011 and 51.026.) 

7.087 * Water-Soluble Acidity (27) * 

Official Final Action 

See 22.053, 10th ed. 

7.088 * Ferrous Salts (2d)— Official Final Action * 

K 3 Fe(CN) 6 spot test. See 7.074, 12th ed. 

7.089 * Copper Salts (25) — Official Final Action * 

K 4 Fe(CN) 6 spot test. See 7.075, 12th ed. 

* Surplus method — see inside front cover. 



7.095 
See 2.112-2.113. 



Determination and Calculation 



Calcium — Official Final Action 
7.096 Method I {30) 

(Applicable to mineral feeds only) 

Weigh 2 g finely ground sample into Si0 2 or porcelain dish 
and ignite in furnace to C-free ash, but avoid fusing. Boil residue 
in 40 mL HCI (1+3) and few drops HN0 3 . Transfer to 250 mL vol. 
flask, cool, dil. to vol., and mix thoroly. Pipet 25 mL clear liq. 
into beaker, dil. to ca 100 mL, and add 2 drops Me red, 2.055(i). 
Add NH4OH (1 + 1 ) dropwise to pH 5.6, as shown by intermediate 
brownish-orange. If overstepped, add HCI (1+3) with dropper 
to orange. Add 2 more drops HCI (1+3). Color should now be 
pink (pH 2.5-3.0), not orange. Dil. to ca 150 mL, bring to boil, 
and slowly add, with const stirring, 10 mL hot satd (4.2%) 
soln of (NH 4 )2C 2 04. If red changes to orange or yellow, add HCI 
(1+3) dropwise until color again changes to pink. Let stand 
overnight for ppt to settle. Filter supernate thru quant, paper, 
gooch, or fritted glass filter (fine Pyrex is preferable), and wash 
ppt thoroly with NH 4 OH (1+50). Place paper or crucible with ppt 
in original beaker, and add mixt. of 125 mL H 2 and 5 mL H 2 S0 4 . 



AOAC Methods (1980) 



Minerals 



137 



Heat to ^70° and titr. with 0.1/V KMn0 4 , 50.025-50.026, to first 
slight pink. Presence of paper may cause color to fade in few 
sec. Correct for blank and calc. % Ca. 



Method II [31) 



7.097 



Preparation of Solution 

{Caution: See 51.026 and 51.028.) 

(a) Weigh 2.5 g sample into 500 or 800 mL Kjeldahl flask. Add 
20-30 mL HN0 3 and boil gently 30-45 min to oxidize all easily 
oxidizable matter. Cool soln somewhat and add 10 mL 70-72% 
HCIO4. Boil very gently, adjusting flame as necessary, until soln 
is colorless or nearly so and dense white fumes appear. Use 
particular care not to boil to dryness (Danger!) at any time. Cool 
slightly, add 50 mL H 2 0, and boil to drive out any remaining 
N0 2 fumes. Cool, dil., filter into 250 mL vol. flask, dil. to vol., 
and mix thoroly. 

(b) Weigh 2.5 g finely ground sample into Si0 2 or porcelain 
dish and ignite as in 7.009. Add 40 mL HCI (1 +3) and few drops 
HNO3 to residue, boil, transfer to 250 mL vol. flask, cool, dil. to 
vol., and mix thoroly. 



7.098 



Determination 



Pipet suitable aliquot of clear soln, 7.097(a) or (b), into beaker, 
dil. to 100 mL, and add 2 drops Me red, 2.055(i). Continue as in 
7.096, beginning "Add NH 4 OH (1+1) dropwise . . ." except use 
0.05/V KMn0 4 for titrn. 

(100 mL is suitable aliquot of sample soln for grain feeds; for 
mineral feeds, 25 mL aliquot may be taken and titrd with 0.1/V 
KMn0 4 . For suitable precision, size of sample, aliquot and concn 
of KMn0 4 must be so adjusted that s*20 mL std KMn0 4 soln is 
used.) 



Soluble Chlorine 
Titration Method (32)- -Official Final Action 



7.099 



Reagents 



(a) Potassium chloride std soln.— 0.001 g CI/mL Recrystallize 
reagent KCI 3 times from H 2 0, dry at 110°, and heat at ca 500° 
to const wt. Dissolve 2.1028 g in H 2 and dil. to 1 L. 

(b) Silver nitrate soln, — Dissolve 5 g AgN0 3 in 1 L H 2 and 
adjust soln so that 1 mL = 1 mL std KCI soln. 

(c) Potassium thiocyanate soln.— Dissolve 2.5 g KSCN in 1 L 
H 2 and adjust so that 1 mL = 1 mL std AgN0 3 soln. Stdze as 
in 50.004. 

(d) Ferric sulfate soln. — Dissolve 60 g Fe 2 (S0 4 ) 3 + Aq. in H 2 
and dil. to 1 L. 

(e) Ferric sulfate indicator. — To filtered 25% soln of Fe 2 (S0 4 ) 3 
+ Aq. add equal vol. HN0 3 . 



7.100 



Determination 



Transfer 3 g sample to 300 mL erlenmeyer. Add 50 mL 
Fe 2 (S0 4 ) 3 soln (accurately measured), swirling flask to prevent 
caking of sample and to facilitate soln of CI. Add 100 mL {also 
accurately measured) NH 4 OH (1 + 19). Swirl flask just enough to 
ensure soln of CI and thoro mixing of soln. (Very little swirling 
is necessary. If soln is agitated by vigorous vertical shaking, 
filtration will be difficult.) Let mixt. settle 10 min. Filter thru dry 
11 cm Whatman No. 41 paper, or equiv. Use 50 mL aliquots ( 1 / 3 
of total) on samples low in CI (0-2% CI) and 25 mL aliquots ( 1 / 6 
of total) on samples high in CI (>2%). For mineral and other 
feeds contg s=10% CI, weigh 1 g and use 15 mL {V10 of total). 

If approx. % CI in sample is not known, take 10 mL aliquot for 
trial titrn. To this add 10 mL HN0 3 and 10 mL Fe 2 (S0 4 ) 3 indicator. 
Dil. to ca 50 mL. Add 0.5 mL KSCN soln and immediately add, 



with stirring, enough AgN0 3 soln to entirely eliminate any 
reddish color. From this titrn calc. vol. AgN0 3 soln necessary to 
ppt all CI in aliquot to be used, adding excess equal to ca 10% 
total vol. necessary, altho somewhat greater excess will not 
affect results. Use min. total of 10 mL 

To sample aliquot in 250 mL beaker add 10 mL HN0 3 and 10 
mL Fe 2 (S0 4 ) 3 indicator (or 20 mL soln contg equal vols of these 
solns). Add, with stirring, calcd vol. AgN0 3 soln. Heat to boiling 
and cool to room temp., stirring enough to coagulate ppt. 
(Cooling may be hastened by immersion of beakers in cold H 2 0.) 
Titr. excess AgN0 3 with KSCN. End point is indicated by first 
appearance of reddish tint that persists 15 sec. For accurate 
work, use ref. soln contg all ingredients except KSCN. End point 
is first change in color. 



Potentiometric Method (33) 
Official Final Action 



7.101 



Apparatus 



Potentiometer. — With Ag-AgCI reference electrode and Ag- 
indicating electrode (Fisher Scientific Co. No. 9-313-216 and 
13-639-122, or equiv.). 



7.102 



Standardization 



Weigh 125 mg dry NaCI into 400 mL beaker. Add 200 mL H 2 
and 1 mL HN0 3 . 

Null potentiometer and titr. NaCI soln with 0.1/V AgN0 3 soln. 
Plot mL AgN0 3 soln against mv or scale readings. Add titrant 
in small enough increments so that voltage end point is obvious. 
Use same end point for samples. 



7.103 



Determination 



(a) Samples containing less than 5% sodium chloride. — 
Weigh 5.844 g sample into 400 mL beaker. Add ca 200 mL H 2 
and 1 mL HN0 3 . Swirl mixt. gently and let stand 10 min for 
complete soln of chlorides. Titr., while stirring, to same voltage 
end point as in stdzn. 

% NaCI - mL 0.1/V AgNO 3 /10. 

(b) Samples containing more than 5% sodium chloride. — 
Weigh 5.844 g sample into 200 mL vol. flask. Add ca 190 mL 
H 2 and 1 mL HN0 3/ dil. to vol. with H 2 0, mix, and let stand 10 
min. Transfer aliquot contg equiv. of ca 125 mg NaCI to 400 mL 
beaker, dil. to ca 200 mL, add 1 mL HN0 3 , and titr. as in (a). 

% NaCI = diln factor x mL 0.1/V AgNO 3 /10. 



Cobalt {34)— Official Final Action 



7.104 



Reagents 

(a) Cobalt std soln.— 0.05 mg Co/mL. Dissolve 0.2385 g 
CoS0 4 .7H 2 (do not dry; use as received) in H 2 and dil. to 1 L. 
Dil., if necessary, to suitable concn to prep, std curve. 

(b) Nitroso-R salt soln.— Dissolve 1 g C 10 H 4 0H.NO(SO 3 Na) 2 in 
H 2 and dil. to 500 mL 

(c) Spekker acid. —M\x 150 mL 85% H 3 P0 4 and 150 mL H 2 S0 4 , 
and dil. to 1 L with H 2 0. 

(d) Sodium acetate soln.— Dissolve 500 g NaOAc.3H 2 in H 2 
and dil. to 1 L with H 2 0. 



7.105 



Preparation of Standard Curve 



To 1, 2, etc., up to 11 mL portions std Co soln in 100 mL vol. 
flasks add 2 mL Spekker acid, 10 mL nitroso-R salt soln, and 10 
mL NaOAc soln. Prep, blank by using 2 mL Spekker acid and 10 
mL NaOAc soln, but omitting nitroso-R salt soln. Bring solns to 
bp on hot plate. Add 5 mL HN0 3 and boil 2=1, but <l min. Cool, 
and dil. to 100 mL. 



138 



7. Animal Feed 



AOAC Methods (1980) 



7.106 



Determination 



{Caution: See 51.026 and 51.059.) 

Ash 2 g sample 2 hr at 600°, transfer to 200 mL vol. flask with 
20 mL HCI and 50 mL H 2 0, boil 5 min, coot, and dil. to vol. Let 
soln settle. Pipet suitable aliquot into small flask. For samples 
contg 0.01-0.2% Co use equiv. of 0.25 g sample. Adjust amt to 
^0.5 mg Co. Soln no longer appears to follow Beer's law above 
this amt. 

Pass brisk current of H 2 S thru soln 10 min. Filter directly into 
100 mL vol. flask thru Whatman No. 40 paper. Wash with ca 50 
mL 7% H 2 S0 4 satd with H 2 S. Add 2 small glass beads and boil 
off H 2 S. (Flasks must be given individual attention, as violent 
bumping may occur.) Shake flasks often. Add 5 mL HN0 3 and 
boil until nitrous fumes no longer appear. (Take care, as vol. of 
soln will be low and bumping and spattering may occur. At first 
indication of this, remove immediately from hot plate.) Small 
amt HN0 3 remaining will not affect result. Cool, add 2 drops 
phthln, and adjust to first faint pink with ca 30% NaOH soln. 
Immediately add 2 mL Spekker acid followed by 10 mL nitroso- 
R salt soln and 10 mL NaOAc soln. Bring to vigorous boil, 
carefully add 5 mL HN0 3 , and boil s?1 but ^2 min. Cool, and dil. 
to vol. 

Compare color with std Co solns in photoelec. colorimeter,' 
using green or No. 54 filter, or in spectrophtr at 540 nm. Read 
color within 2 hr. Report % Co to third decimal place. 



Copper [35) — Official Final Action 

7.107 Preparation of Standard Curve 

Dissolve 1.9645 g CuS0 4 .5H 2 in H 2 and dil. to 500 mL. (1 
mL = 1 mg Cu.) Use from 1 to 10 mL of this soln to prep, set of 
stds in 100 mL Pyrex g-s vol. flasks. Add 4 mL HCI, dil. to 50 mL, 
add 5 mL tetraethylenepentamine, dil. to vol. with H 2 0, stopper, 
and mix thoroly. Prep, blank, using all reagents except Cu. Filter 
blank and stds before reading color as in 7.108. 



7.108 



Determination 



Prep, sample soln as in 7.106, using 8 g sample. Pipet 50 mL 
aliquot into 100 mL Pyrex g-s vol. flask, add 5 mL tetraethyle- 
nepentamine, dil. to vol. with H 2 0, and mix thoroly. Filter, and 
compare colors within 30 min in photoelec. colorimeter (red or 
No. 66 filter) or read in spectrophtr at 620 nm. Report % Cu to 
third decimal place. 



Fluorine — Official Final Action 

7. 1 09 Color/metric Method 
See 25.049-25.055, especially 25.053. 

Ion Selective Electrode Method [36) 

7.110 Apparatus 

(a) Electrodes. — Fluoride ion selective electrode (Model 94- 
09, Orion Research Inc., or equiv.) and single junction calomel 
ref. electrode, plastic sleeve-type (Model 90-01, Orion Research 
Inc., or equiv.). 

(b) Magnetic stirrer. — With 4 cm (IV2") Teflon-coated stirring 
bar. Use mat to insulate sample from motor heat. 

(c) pH meter. — Corning digital Model 112 (Corning Scientific 
Instruments, Medfield, MA 02052, or equiv.). 



7.111 Reagents 

{Deionized H 2 may be used.) 

(a) Sodium acetate soin—3M. Dissolve 408 g NaOAc.3H 2 
with H 2 in 1 L vol. flask. When soln warms to room temp., dil. 
to vol. with H 2 0. Adjust to pH 7.0 with few drops HOAc. 

(b) Sodium citrate soln. — 1.32/W. Dissolve 222 g Na 
citrate.2H 2 with ca 250 mL H 2 in 1 L vol. flask. Add 28 mL 
HCIO4, dil. to vol., and mix. 

(c) Fluoride std solns. — (7) Stock soln. — 500 ppm. Accurately 
weigh 1.105 g NaF (reagent grade, dried 4 hr at 100°) into 1 L 
vol. flask. Dissolve and dil. to vol. with H 2 0, and mix. Store in 
plastic bottle. (2) Intermediate soln A— 100 ppm. Pipet 20 mL 
stock soln into 100 mL vol. flask, dil. to vol. with H 2 0, and mix. 
{3) Intermediate soln II. — 10 ppm. Pipet 2 mL stock soln into 100 
mL vol. flask, dil. to vol. with H 2 0, and mix. {4) Working solns. — 
Pipet 3, 5, and 10 mL intermediate soln II and 5 and 10 mL 
intermediate soln I into five 100 mL vol. flasks to prep. 0.3, 0.5, 
1.0, 5.0, and 10 ppm F working solns, resp. To each add 10.0 mL 
1/V HCI, 25.0 mL NaOAc.3H 2 soln, (a), and 25.0 mL Na citrate 
soln, (b). Dil. to vol. with H 2 and mix. 

7.112 Preparation of Sample 

Accurately weigh well mixed sample contg ca 400 jug F into 
200 mL vol. flask. Pipet in 20 mL 1/V HCI and stir 20 min at high 
speed on mag. stirrer. Add 50.0 mL NaOAc soln, (a), and 50.0 
mL Na citrate soln, (b), to dissolved sample. Dil. to vol. with H 2 
and mix. 



7.113 



Determination 



Connect F and ref. electrodes to pH meter, place electrodes 
in low concn F soln, and warm up pH meter. Pour 50-70 mL std 
and corresponding sample solns into sep. 100 mL beakers. Place 
electrodes in each soln and while stirring with mag. stirrer at 
const rate, read mv of std and unknown solns. Rinse and 
blot off electrodes and stirring bar between solns. Construct std 
curve on 3 cycle semilogarithmic paper. Read ppm F of sample 
soln from std curve. 

% F = ppm F x mL sample soln x 10~ 6 x 100/g sample 

Iodine in Mineral Mixed Feeds — Official Final Action 

7.114 * Knapheide-Lamb Method [37) * 
See 22.084-22.086, 10th ed. 

7.115 Elmslie-Caldwell Method [38) 

(Not applicable to iodized mineral feeds contg 
little or no org. matter. Caution: See 51.047.) 

Place sample contg 3-4 mg I in 200-300 mL Ni dish. Add ca 
5 g Na 2 C0 3 , 5 mL NaOH soln (1 + 1), and 10 mL alcohol, taking 
care that entire sample is moist. Dry at ca 100° to prevent 
spattering upon subsequent heating (30 min is usually enough). 

Place dish and contents in furnace heated to 500° and keep at 
that temp. 15 min. (Ignition of sample at 500° appears to be 
necessary only to carbonize any sol. org. matter that would be 
oxidized by Br-H 2 if not so treated. Temp. >500° may be used 
if necessary.) Cool, add 25 mL H 2 0, cover dish with watch glass, 
and boil gently 10 min. Filter thru 18 cm paper and wash with 
boiling H 2 0, catching filtrate and washings in 600 mL beaker 
(soln should total ca 300 mL). Neutze to Me orange with 85% 
H3PO4 and add 1 mL excess. 



* Surplus method— see inside front cover. 



AOAC Methods (1980) 



Microscopy 



139 



Add excess Br-H 2 and boil soln gently until colorless, and 
then 5 min longer. Add few crystals salicylic acid and cool soln 
to ca 20°. Add 1 mL 85% H 3 P0 4 and ca 0.5 g Kl, and titr. I with 
0.005/V Na 2 S 2 3 , adding starch soln when liberated I color is 
nearly gone. 1 mL 0.005/V Na 2 S 2 3 = 0.1058 mg I. 

Acid-Soluble Manganese [39) 
Official Final Action 

7.116 Reagent 

Potassium permanganate std soln. — 500 ppm Mn. Prep, and 
stdze as in 50.025-50.026, except use 1.4383 g KMn0 4 and 0.12 
g Na oxalate. Transfer aliquot contg 20 mg Mn to beaker. Add 
100 mL H 2 0, 15 mL H 3 P0 4 , and 0.3 g KI0 4 , and heat to bp. Cool, 
and dil. to 1 L. Protect from light. Dil. this soln contg 20 ppm Mn 
with H 2 (previously boiled with 0.3 g KI0 4 /L) to make convenient 
working stds in range of concns to be compared. 



7.117 



Determination 



{Caution: See 51.026 and 51.030.) 



Ash weighed sample, 5-15 g, at dull red heat (ca 600°) in 
porcelain dish. Cool, and add 5 mL H 2 S0 4 and 5 mL HN0 3 to ash 
in dish or to ash transferred to beaker with 20-30 mL H 2 0. Evap. 
to white fumes. If C is not completely destroyed, add addnl 
portions HN0 3 , boiling after each addn. Cool slightly, transfer 
to 50 or 100 mL vol. flask, and add vol. dil. H 3 P0 4 soln (8+92) 
equal to V 2 vol. of flask (25 or 50 mL). Cool, dil. to vol., mix, and 
filter or let stand until clear. 

If 50 mL flask was used, pipet 25 mL clear soln into beaker or 
50 or 100 mL vol. flask and add 15 mL H 2 0. If 100 mL flask was 
used, pipet 50 mL into beaker or 100 mL flask and add 30 mL 
H 2 0. Heat nearly to bp, and with stirring or swirling add 0.3 g 
KI0 4 for each 15 mg Mn present. Keep 30-60 min at 90-100°, or 
until color development is complete. Cool, dil. to measured vol. 
of 50 or 100 mL, and mix. Compare with std KMn0 4 soln in 
photoelec. colorimeter or in spectrophtr at 530 nm. Calc. ppm 
Mn. 



<5%; 0.2 g for 5-20%; 0.1 g for >20%. Add 5-10 mL HN0 3 , 
depending on method of soln (or equiv. in NH 4 N0 3 ); then add 
NH 4 OH until ppt that forms dissolves only slowly on vigorous 
stirring, dil. to 75-100 mL, and adjust to 25-30°. If sample does 
not give ppt with NH 4 OH as test of neutzn, make soln slightly 
alk. to litmus paper with NH 4 OH and then slightly acid with HN0 3 
(1+3). Add 20-25 mL acidified molybdate soln for P 2 5 content 
<5%; 30-35 mL for 5-20%; and enough acidified molybdate 
soln to ensure complete pptn for >20%. Shake or stir mech. 30 
min at room temp.; decant at once thru filter and wash ppt 
twice by decanting with 25-30 mL portions H 2 0, agitating 
thoroly and allowing to settle. Transfer ppt to filter and wash 
with cold H 2 until filtrate from 2 fillings of filter yields pink 
color on adding phthln and 1 drop of the std alkali. Transfer ppt 
and filter to beaker or pptg vessel, dissolve ppt in small excess 
of the std alkali, add few drops of phthln, and titr. with std "acid. 
Report as % P. 



Photometric Method [41) — Official Final Action 

7. 1 20 Apparatus 

Spectrophotometer.— Capable of isolating 400 nm band and 
accepting ==15 mm diam. cells. 

7.121 Reagents 

(a) Molybdovanadate reagent.— Prep, as in 2.022(a), except 
add only 250 mL 70% HCI0 4 to NH 4 V0 3 soln. 

(b) Phosphorus std solns.—{1) Stock soln.— 2 mg P/mL Dis- 
solve 8.788 g KH 2 P0 4 in H 2 and dil. to 1 L. {2) Working soln.— 
0.1 mg P/mL. Dil. 50 mL stock soln to 1 L. 

7.122 Preparation of Standard Curve 

Transfer aliquots of working std soln contg 0.5, 0.8, 1.0, and 
1.5 mg P to 100 mL vol. flasks. Treat as in 7.123, beginning 
"Add 20 mL molybdovanadate reagent, . . ." Prep, std curve by 
plotting mg P against %T on semilog paper. 



Phosphorus 

Alkali metric Ammonium Molybdophosphate 
Method [40)— Official Final Action 

7.118 Reagents 

(a) Molybdate soln. — Dissolve 100 g Mo0 3 in mixt. of 144 mL 
NH 4 OH and 271 mL H 2 0. Cool, and slowly pour soln, stirring 
constantly, into cool mixt. of 489 mL HN0 3 and 1148 mL H 2 0. 
Keep final mixt. in warm place several days or until portion 
heated to 40° deposits no yellow ppt. Decant soln from any 
sediment and keep in g-s vessels. 

(b) Acidified molybdate soln. — To 100 mL molybdate soln, 
(a), add 5 mL HN0 3 . Filter immediately before use. 

(c) Sodium hydroxide std $oln.—0\\. 324.03 mL 1/V alkali, 
carbonate-free, 50.032~50.036, to 1 L. (100 mL of this soln should 
neutze 32.40 mL 1/V acid; 1 mL = 1 mg or 1% P 2 5 on basis of 
0.1 g sample.) (Since burets in const use may become so 
corroded as to increase their capacity, test them at least an- 
nually.) 

(d) Std acid soln. — Prep, soln of HCI orof HN0 3 , corresponding 
to concn of (c) or to !/ 2 this concn, and stdze by titrn against (c), 
using phthln. 



7.123 



Determination 



7.119 



Determination 



Prep, sample soln as in 7.097(a). Pipet, into beaker or flask, 
aliquot corresponding to 0.4 g sample for P 2 5 content of sample 



Ash 2 g sample, in 150 mL beaker, 4 hr at 600°. Cool, add 40 
mL HCI (1+3) and several drops HN0 3 , and bring to bp. Cool, 
transfer to 200 mL vol. flask, and dil. to vol. with H 2 0. Filter, and 
place aliquot contg 0.5-1.5 mg P in 100 mL vol. flask. Add 20 
mL molybdovanadate reagent, dil. to vol. with H 2 0, and mix 
well. Let stand 10 min; then read %T at 400 nm against 0.5 mg 
std set at 100% 7". (Use =S15 mm diam. cells.) Det. mg P from 
std curve. 

% P = mg P in aliquot/(g sample in aliquot x 10). 



Basic Feed Microscopy {42)— Official Final Action 
7.124 Apparatus 

(a) Magnifier-fluorescent illuminator with desk base, 3x, or 
reading glass. 

(b) Microscopes and illuminator.— See 44.002(n) and (q). Fol- 
lowing are preferred: 

(7) Widefield stereoscopic microscope.— With arm rests, flat 
stage (remove spring holders), optional substage illumination, 
inclined eyepiece, and lenses to magnify ca 7-30 x, 15x opti- 
mum. 

(2) Compound microscope. — With mech. stage, substage con- 
denser, inclined binocular eyepiece, 3 position rotating nose- 
piece, lenses to magnify ca 36-400X, 120x optimum. 



140 



7. Animal Feed 



AOAC Methods (1980) 



{3) Microscope illuminator. — With iris diaphragm; movable 
stand holder with rod to permit adjusting light source as to ht 
and angle for substage or direct over-stage lighting; able to hold 
2 blue glass filters or 1 blue and 1 ground glass; 60-100 watt 
bulb. 

(c) Sieves. — Nest of 5" No. 10, 20, 40, 60, 80, and bottom pan. 

(d) Stages. — Dark Co glass plates 4 x 4" (Fisher Scientific Co. 
No. 13-735); or blue paper and microscope slides. 

(e) Spot plates. — Black and white. 

(f) Forceps. — Fine pointed, curved. If necessary, bend and 
grind on emery wheel for good contact of points. 

(g) Dropping bottles. — Amber, 30 mL, as reagent dispensers. 
(h) Micro-spatula; micro-stirring rods made by drawing out 

glass rods; spoon. 



that permits identification of components when compared to 
std feed ingredients. 



7.125 



Reagents 



(a) Chloroform. — Tech. Recover by filtration and distn. 

(b) Acetone. — Tech. 

(c) Acetone, dilute. — Dil. 75 mL acetone with 25 ml_ H 2 0. 

(d) Dilute hydrochloric acid '.—Dil. 1 vol. HCI with 1 vol. H 2 0. 

(e) Dilute sulfuric acid.—DW. 1 vol. H 2 S0 4 with 1 vol. H 2 0. 

(f) Iodine soln.— Dissolve 0.75 g Kl and 0.1 g I in 30 mL H 2 
and add 0.5 mL HCI. Store in amber dropping bottle. 

(g) Mil ion reagent. — Dissolve, by gently warming, 1 part by 
wt Hg in 2 parts by wt HN0 3 . Dil. with 2 vols H 2 0. Let mixt. stand 
overnight and decant supernate. Soln contains Hg(N0 3 ) 2/ HgN0 3 , 
HN0 3 , and some HN0 2 . Store in g-s bottle. {Caution: See 51.079.) 

(h) Moiybdate soln.— Add 100 mL 10% NH 4 N0 3 soln to 400 
mL moiybdate soln, 7.118(a). Use only clear supernate to fill 30 
mL amber dropping bottle. Discard and refill when crystn occurs. 

(i) Mountant I. — Dissolve 10 g chloral hydrate in 10 mL H 2 
and add 10 mL glycerol. Store in amber dropping bottle. 

(j) Mountant II.— Dissolve 160 g chloral hydrate in 100mLH 2 O 
and add 10 mL HCI. 

(k) Silver nitrate soln.— 10%. Dissolve 10 g AgN0 3 in 100 mL 
H 2 0. 



7.126 



Standards 



(a) Feed ingredients. — Collect ingredients used in grain and 
stock feeds known to conform to definitions of Association of 
American Feed Control Officials as stds. Store in 4 02 bottles. 
To control insects, add ca 1 mL CS 2 , and stopper. Become 
thoroiy familiar with structural appearance of stds before and 
after treatment with org. solvs. 

(b) Weed seeds. — Collect common weed seeds occurring in 
grains. Most may be found in foreign material obtained after 
sieving com. whole grains with U.S. Grain Testing Sieve having 
5 / 64 " (2.5 mm) triangular holes. Identify from illustration in 
"Identification of Crop and Weed Seeds" (USDA Handbook 219 
(1963), Government Printing Office, Washington, DC 20402). 
Store in numbered vials. Become familiar with those weed 
seeds designated as prohibited and restricted noxious under 
state laws of individual concern. {See "State Noxious-Weed 
Seed Requirements Recognized in the Administration of the 
Federal Seed Act" (USDA, Agricultural Marketing Service, Grain 
Div., Hyattsviile, MD 20782).) 



Identification of Vegetable Tissues 



7.127 



Principle 



Feeds are fractionated according to particle size and cleared 
where necessary for clear observation; conglomerates are dis- 
integrated into constituents and fractions arranged on stage 
suitable for microscopic examination at lowest magnification 



7.128 



General Methods 



(a) Scratch feeds. — Spread representative portion of sample 
on white paper and examine under magnifier-fluorescent illu- 
minator at 3x or with reading glass. Identify grains and weed 
seeds; note other foreign material, heat- and insect-damaged 
particles, live insects, and rodent excreta; examine for smut, 
ergot, and mold (''Grain Inspection Manual," USDA). 

(b) Mashes comparatively free from adhering fine particles. — 
(7) Low power microscopy . — Arrange in nest form 3 sieves that 
will adequately fractionate feed according to particle size. Gen- 
erally, for cattle feeds use No. 10, 20, and 40; for poultry feeds, 
No. 20, 40, and 60. Include bottom pan. Add ca 10 g unground 
feed (plastic tablespoon makes convenient scoop) to nest, and 
sieve thoroiy. With spatula, spread portion from each sieve on 
4 x 4" Co glass stage and place under stereoscopic microscope. 
(Blue paper may also be used as stage.) Arrange illuminator 
above and near stage so light strikes sample at angle of ca 45° 
for shadow contrast. Adjust magnification (ca 15x optimum), 
illumination, and light filters to individual preference for clear 
observation. Blue light or northern daylight is preferred. Examine 
each fraction on stage sep. and systematically. Observe feed 
particles, continually probing, turning, and testing resistance to 
pressure with forceps. Note particle size, shape, color, resistance 
to pressure, texture, odor, and major structural features. Com- 
pare with stds. If desired, transfer individual particles with 
forceps to second glass plate for direct comparison with cor- 
responding tissues from stds. Likewise transfer and break up 
conglomerates by gentle pressure with flat end of forceps. Make 
list of observed ingredients. Neglect trace grains which may be 
normal inpurities in major grains. (Consult "Official Grain Stand- 
ards of the United States," USDA, for amts of "other grains" 
permissible as impurities in whole grains.) 

(2) High power microscopy. — Lower illuminator and select 
filters so adequate blue light is reflected thru substage condenser 
of high power microscope. With microspatula, transfer little of 
fine sievings from bottom sieve and pan to slide, add 2 drops 
mountant I, stir, and disperse with microstirring rod. Examine 
microscopically (120x optimum). Compare histologically with 
stds. Remove slide, add 1 drop I soln, stir, and re-examine. 
Starch cells are stained pale blue to black; yeasts and other 
protein cells, pale yellow to brown. If further tissue clarification 
is desired, boil little of same fine sievings 1 min with ca 5 mL 
mountant II. Cool, transfer drop or 2 of bottom settlings to slide, 
cover, and examine microscopically. 

(c) Oily feeds or those containing large particles obscured by 
adhering fine particles. — (Most poultry feeds and unknowns are 
best examined by this technic.) Place ca 10 g unground feed in 
100 mL tall-form beaker and nearly fill with CHCI 3 (hood). Stir 
briefly and let settle ca 1 min. With spoon, transfer floating (org.) 
material to 3.5" (9 cm) cover glass, drain, and dry on steam 
bath. Sieve, and proceed as in (b). If desired, filter, dry, suspend 
fine particles in CHCI 3 , and examine microscopically (rarely 
necessary). 

(d) Feeds in which molasses has caused lumpiness and 
otherwise obscured vision. — Place ca 10 g unground feed in 100 
mL tall-form beaker. Add 75 mL 75% acetone, stir few min to 
dissolve molasses, and let settle. Carefully decant and repeat 
extn. Wash residue twice with acetone by decantation, dry on 
steam bath, sieve, and proceed as in (b). 

(e) Pellets or crumbles. — Gently grind few pellets at time in 
mortar with pestle with enough pressure to sep. pellet into its 
constituents, but not to break up constituents themselves. Sieve 
first grind thru No. 20 sieve and return particles remaining on 



AOAC Methods (1980) 



Preservatives 



141 



sieve to mortar for further grinding. Depending on nature of 
pellet, proceed with ground material as in (b), (c), or (d). 



identification of Animal Tissues and 
Major Mineral Constituents 



7.129 



Principle 



Feeds contg animal tissues and minerals when suspended in 
CHCI 3 readily sep. into 2 fractions: (/) Org. fraction which floats, 
consisting of muscle fibers, connective tissue, dried ground 
organs, feather remains, hoof and horn particles, etc. from either 
animal or marine products, plus all vegetable tissues. (2) Mineral 
fraction which sinks, consisting of bones, fish scales, teeth, and 
minerals. 



7.130 



Preparation of Sample 



Perform CHCI 3 flotation sepn as in 7.128(c). Collect floating 
material and dry on steam bath. Decant CHCl 3 , collect mineral 
fraction, and dry on steam bath. 

7.131 Identification of Animal Tissue 

Examine dried floating material as in 7.128(b). 

7.132 Identification of Major Mineral Constituents 

Place dried mineral fraction on nest of No. 40, 60, and 80 
sieves and bottom pan. Sieve and place the 4 fractions in sep. 
groups on same Co glass plate or blue paper stage. Examine 
under stereoscopic microscope at ca 15x. Animal and fish 
bones, fish scales, and mollusc shells are generally recogniz- 
able. Salt usually occurs in cubes which may be dyed. Calcite 
form of limestone occurs as rhombohedrons. 



7.133 



Confirmatory Tests 



With forceps, place unknown particle on glass plate and break 
up by applying gentle pressure with flat surface. Working under 
stereoscopic microscope, sep. particles ca 2.5 cm and place 
beside each a fractional drop of reagent solns listed by touching 
end of dropper to plate. Push particle into liq. with microstirring 
rod and observe what occurs at interface. Follow order given 
until pos. identification is obtained. If preferred, perform tests 
in black spot plate. 

(a) Silver nitrate soln. — (7) Crystal immediately turns chalk 
white and slowly expands: chloride, probably salt. (2) Crystal 
turns yellow and yellow needles begin to grow: mono- or dibasic 
phosphate, generally dicalcium phosphate. (5) Sparingly sol. 
white needles form (Ag 2 S0 4 ): sulfate, Mn-MgS0 4 . (4) Particles 
slowly darken: bone. 

(b) Dilute hydrochloric acid. — (/) Vigorous effervescence: 
CaC0 3 . (2) Mild effervescence or none: make following tests. 

(c) Molybdate soln. — Formation of minute yellow crystals at 
some distance from particle: tricalcium phosphate, either bone 
or rock phosphate. (All phosphates react, but mono- and dibasic 
phosphates have been identified with AgN0 3 .) 

(d) Mil Ion reagent. — (7) Disintegrated particles mostly float, 
turn pink to red (protein), and fade in ca 5 min: bone phosphate. 
(2) Particles appear to swell and disintegrate but remain on 
bottom: defluorinated rock phosphate. {3) Particles merely dis- 
integrate slowly: rock phosphate. 

(e) Dilute sulfuric acid. — Long, thin white needles slowly form 
on addn of drop of H 2 S0 4 (1 + 1) to HCI (1 + 1) soln of particle: 
confirms Ca. 

7.134 Identification of Furazolidone, Tylosin, and Zoalene 
See 42.001-42.004. 



PRESERVATIVES 

Ethoxyquin (1,2-Dihydro-6-ethoxy-2,2,4- 
trimethylquinoline) (45)— Official Final Action 



7.135 Reagents and Apparatus 

(a) Quinine sulfate reference soln.— 1 m9/tiL 0.1 N H 2 S0 4 . 
Dissolve 0.100 g quinine sulfate USP in 1 L 0.1/V H 2 S0 4 . Dil. 10 
mL aliquot of this soln to 1 L with 0.1/V H 2 S0 4 . Use to calibrate 
photofluorometer. 

(b) Ethoxyquin std solns. —Add 100.0 mg liq. ethoxyquin to 
100 mL vol. flask and dil. to vol. with pet ether {Soln A). Dil. 5 
mL Soln A to 100 mL with pet ether {Soln B, 50 ptg/mL). Dil. 5 
mL Soln B to 100 mL with pet ether {Soln C, 2.5 pig/mL). Dil. 10 
ml Soln Cto20 mL with pet ether (1.25 fig/ ml) and 5mLto 25 
mL (0.50 ju-g/mL). 

(c) Photofluorometer. — Equipped with primary filter passing 
365 nm Hg line (Coming Glass Works No. 5874 (CS7-39), or 
equiv.) and secondary filter passing 420-500 nm (Corning Glass 
Works 3389 + 5543 + 4784, half stock thickness, or equiv.). 



7.136 



Preparation of Standard Curve 



Adjust photofluorometer to read with pet ether and 100 with 
quinine sulfate ref. soln. Obtain fluorescence readings for 
ethoxyquin std solns contg 0-2.5 fig/mL Plot readings against 
fjug ethoxyquin/mL on linear paper. 



7.137 



Determination 



Place 10±0.1 g finely ground sample in 100 mL beaker and 
slurry with 50 mL MeOH. Stir and let stand 10 min. Decant thru 
plug of glass wool into 250 mL vol. flask. Reslurry residue with 
two 50 mL portions MeOH, decant, and filter, combining all 
filtrates. Dil. to vol. with MeOH. Transfer 25 mL aliquot to 250 
mL separator, add 100 mL H 2 0, and mix well. Add 50 mL pet 
ether, stopper, and shake moderately 1 min. Let stand few min 
to sep. (If emulsion forms, add ca 100 mg NaCI crystals. After 
emulsion breaks, drain aq. lower layer into 250 mL beaker.) 
Transfer pet ether layer to second 250 mL separator, return aq. 
layer to first separator, and re-ext with two 25 mL portions pet 
ether. 

Add 50 mL H 2 to combined pet ether exts in separator, 
stopper, and shake moderately, Let sep., drain lower aq. layer, 
and discard. Transfer pet ether layer to 100 mL vol. flask, and 
dil. to vol. with pet ether. Adjust photofluorometer as above 
and det. fluorescence readings. Obtain jug ethoxyquin/mL from 
std curve. Ppm ethoxyquin = 100 x yxg/mL. 

If untreated feed is available, prep, std curve from series of 
samples contg 0-250 jug ethoxyquin/ 10 g and carried thru detn. 

7.138 Drugs in Feeds 

See Chapter 42. 

7.139 Molasses and Molasses Products 

See Chapter 31. 

7.140 Cyanogenetic Glucosides 
See 26.134. 

7.141 Hydrocyanic Acid Formed by Hydrolysis of 
Glucosides in Beans 

See 26.135-26.136. 



142 



7. Animal Feed 



AOAC Methods (1980) 



SELECTED REFERENCES 

(7) J AOAC 33, 424(1950); 41, 223(1958); 48, 658(1965). 

(2) JAOAC 17, 68(1934). 

(3) JAOAC 8, 295(1925); 9, 30(1926). 

(4) USDA Bur. Chem. Bull. 122, p. 219; 132, p. 150. 

(5) JAOAC 13, 173(1930); 14, 152(1931); 17, 178(1934); 18, 
80(1935). 

(6) JAOAC 36, 213(1953); 37, 253(1954). 

(7) JAOAC 25, 857(1942); 26, 220(1943). 

(8) JAOAC 18,81,369(1935). 

(9) JAOAC 37, 241(1954); 38, 56(1955). 
(10) JAOAC 51, 766(1968). 

(77) JAOAC 59, 141(1976). 

{12) JAOAC 59, 134(1976); 62, 290(1979). 

(73) JAOAC 24, 867(1941); 25, 874(1942); 27, 494(1944). 

{14) JAOAC 50,56(1967). 

(75) JAOAC 51, 763(1968). 

{16) J. Agric. Food Chem. 3, 159(1955); JAOAC 40, 606(1957); 

41, 233(1958); 42, 231(1959); 43, 320(1960); 54, 669(1971); 

55,702(1972). 
(77) JAOAC 37, 250(1954); 38, 225(1955); 59, 1218(1976). 
(75) JAOAC 15, 524(1932); 17, 190(1934); 18, 351(1935); 28, 

80(1945). 
{19) JAOAC 42, 222(1959); 43, 335(1960); 44, 567(1961); 45, 

578(1962). 
{20) JAOAC 61, 154(1978). 
{21) JAOAC 46, 829(1963); 56, 781(1973). 
{22) JAOAC 57,382(1974). 



{23) USDA Bur. Chem. Circ. 71; JAOAC 41, 276(1958); 42, 

39(1959). 
{24) J. Agr. Research 23, 995(1923); JAOAC 9, 31 (1926). 

(25) JAOAC 21, 595(1938); 23, 656(1940). 

(26) J. Landw. 48, 357(1900); 49, 7(1901). 

(27) USDA Bur. Chem. Bull. 137, p. 152; JAOAC 30, 594(1947). 

(28) JAOAC 14, 142(1931); 15, 77(1932); 23, 86(1940); 28, 
80(1945). 

(29) JAOAC 51, 776(1968); 59, 937(1976). 

(30) JAOAC 10, 177(1927); 19, 93, 574(1936); 28, 80(1945). 

(31) Ind. Eng. Chem., Anal. Ed. 7, 116, 167(1935); JAOAC 30, 
606(1947); 31, 98(1948); 32, 650(1949); 33, 162(1950); 34, 
563(1951). 

(32) JAOAC 26, 87(1943); 28, 80(1945). 

(33) JAOAC 52, 607(1969) 

(34) JAOAC 35, 559(1952). 

(35) Anal. Chem. 19, 325(1947); JAOAC 37, 246 (1954); 38, 
222(1955). 

(36) JAOAC 58, 477(1975). 

(37) JAOAC 17, 67, 173(1934); 18, 335(1935); 38, 96(1955). 

(38) JAOAC 18, 338(1935); 21, 596(1938); 23, 688(1940); 33, 
83(1950). 

(39) J. Am. Chem. Soc. 39, 2366(1917); G. Frederick Smith 
Chemical Co. Pub. 209, 5th ed. (1950); JAOAC 22, 78, 
673(1939); 24, 865(1941); 25, 892(1942). 

(40) USDA Div. Chem. Bull. 56, 36(1898); JAOAC 47, 420(1964). 

(41) JAOAC 48, 654(1965). 

(42) JAOAC 47, 504(1964). 

(43) JAOAC 44, 560(1961); 46, 306(1963); 47, 512 (1964). 



8. Baking Powders and Baking Chemicals 



8.001 Preparation of Sample — Official Final Action 

Remove entire sample from package, pass thru No. 20 sieve, 
and mix thoroly. 

Total Carbon Dioxide (7) — Official Final Action 

(Applicable to baking powders contg added CaC0 3 ) 

8.002 Reagent 

Displacement so/n. — Dissolve 100 g NaCI or Na 2 SO 4 .10H 2 O in 
350 ml_ H 2 0. Add ca 1 g NaHC0 3 and 2 mi_ Me orange, 5.014(e), 
and then enough H 2 S0 4 (1+5) or HCI (1+2) to make just acid 
(decided pink). Stir until all C0 2 is removed. This soln is used 
in gas-measuring tube and leveling bulb and seldom needs 
replacement. 

8.003 Apparatus 

Chittick apparatus. — Fig. 8:01. Connect decomposition flask, 
A, by glass T-tube, B, provided with stopcock, C, to graduated 
gas-measuring tube, D, connected in turn with leveling bulb, E. 
For A always use 250 mL wide-mouth extn flask of Pyrex or 




other resistant glass fitted with 2-hoie rubber stopper, thru one 
hole of which passes extended tip of 25 mL buret, F, and thru 
other, glass tube of same diam. as connecting T-tube. Use buret 
graduated in mL at 20°, numbered at 5 mL intervals, and fitted 
with extra-long tip bent to pass thru rubber stopper. Connect 
glass tube leading from decomposition flask to T-tube with 
rubber tubing to permit rotation of flask. Use gas-measuring 
tube graduated in mL at 20° with mark at point 25 mL below 
top marking to allow for graduating upward from to 25 mL 
and downward from to 200 mL. Connect gas-measuring tube 
to ca 300 mL leveling bulb with long rubber tube. 
(Available from Sargent-Welch Scientific Co.) 



8.004 



Determination (2) 



FIG. 8:01 — Chittick apparatus for g as o metric determination of carbon 
dioxide 



Weigh 1.7 g prepd sample, 8.001, into flask A, and connect 
flask with app,, Fig. 8:01. Open stopcock C, and using leveling 
bulb E, bring displacement soln to 10 mL graduation above 
mark. (This 10 mL is practically equal to vol. of acid to be used 
in decomposition.) Let app. stand 1-2 min fortemp. and pressure 
within app. to come to room conditions. 

Close stopcock, lower leveling bulb somewhat to reduce 
pressure within app., and slowly add 10 mL H 2 S0 4 {1 +5) or HCI 
(1+2) to decomposition flask from buret F. To prevent escape 
of liberated CO z thru acid buret into air, at all times during 
decomposition keep displacement soln at level lower in leveling 
bulb than that in gas-measuring tube. Rotate and then vigorously 
agitate flask to mix contents intimately. Let stand 5 min to 
secure equilibrium. Equalize pressure in measuring tube, using 
leveling bulb, and read vol. of gas in tube. Observe temp, of air 
surrounding app. and also barometric pressure, and multiply 
mL gas evolved by factor given in table, 52.007, for this temp, 
and pressure. 

% C0 2 by wt = corrected reading/ 10. 



8.005 Residual Carbon Dioxide [3) — Official Final Action 

(a) After drying on water bath. — Place 1.7 g baking powder 
in clean, dry, 250 mL wide-mouth Soxhlet extn flask, A, Fig. 
8:01. Add 20 mL H 2 0. Put flask on cover of H 2 bath (single or 
multiple) in which boiling H 2 is kept at const level of 5 cm 
below top of bath. (H 2 in bath must boil vigorously all thru 
detn. Opening in cover of bath must be 7.5 cm diam. to prevent 
flask from touching H 2 0.) Evap. contents of flask until no 
moisture is visible in residue or inside surface of flask. (Sample 
should be completely dry in 1.5-2 hr.) Leave flask on H 2 bath 
2 hr more. Add 10 mL H 2 0, and let stand until flask is at room 
temp, (ca 1 hr). 

Det. C0 2 with Chittick app. as in 8.004, using correction factors 
in 52.007. Shake flask vigorously until further shaking produces 
no increase in reading. 

(b) After drying in oven. — Place 1.7 g sample in clean, dry, 
250 mL wide-mouth Soxhlet extn flask, A, Fig. 8:01. Tap flask 
to spread sample evenly on bottom. Add 10 mL H 2 with pipet. 
Stir with glass rod to break up powder that may have caked on 
bottom of flask. Wash down stirring rod and sides of flask with 
10 mL H 2 0. Place flask on shelf near center of air oven set at 
100±2°, and evap. to dryness. After 5 hr, remove from oven, 
add 10 mL H 2 0, and cool to same temp, as air surrounding 
Chittick app., 8.003. Det. C0 2 in residue with Chittick app., using 
correction factors in 52.007. Shake flask vigorously until further 
shaking produces no increase in reading. 



143 



144 



8. Baking Powders and Baking Chemicals 



AOAC Methods (1980) 



8.006 Available Carbon Dioxide — Official Final Action 

(Applicable to baking powders contg added CaC0 3 ) 
Subtract residual C0 2 , 8.005, from total C0 2 , 8.004. 

Neutralizing Value — Official Final Action 

8.007 Of Acid-Reacting Materials Other Than Phosphates 

Dissolve 1 g sample in hot H 2 and titr. with 0.2/V NaOH, 
using phthln. Express result as parts NaHC0 3 equiv. to 100 parts 
of the acid-reacting material. 

8.008 Of Monocafcium Phosphate {4) 

Weigh 0.84 g sample into 375 mL casserole. Add 24 mL cold 
H 2 and, after stirring for moment, add 90.0 mL 0.1/V NaOH. 
Bring suspension to bp in exactly 2 min, and boil 1 min. While 
soln is still boiling hot, add 1 drop phthln, and back-titr. with 
0.2/V HCI until all pink disappears. Boil soln 1 min, and again 
add 0.2/V HCI until pink just disappears. 

90 - (mL 0.2/V HCI x 2) = neutzg value, parts NaHC0 3 equiv. 
to 100 parts of the phosphate. 

8.009 Of Anhydrous Monocafcium Phosphate [4) 

Use 100 mL 0.1/V NaOH and stir intermittently 5 min before 
bringing to bp. Proceed as in 8.008, 

8.010 Of Sodium A cid Pyrophosphate {4 ) 

Weigh 0.84 g sample and 20 g NaCI into 375 mL casserole, 
and slowly add 25 mL H 2 while stirring. Stir and crush with 
flat-end rod 3-5 min. Add 90.0 mL 0.1/V NaOH and 1 drop phthln, 
and titr. with 0.2/V HCI until pink disappears. If "starch filled" or 
50% neutzg strength pyrophosphate is being titrd, use 70.0 mL 
0.1/V NaOH. 

Vol. (mL) 0.1/V NaOH - (mL 0.2/V HCI x 2) = neutzg value, 
parts NaHC0 3 equiv. to 100 parts Na acid pyrophosphate. 



8.013 Tartaric Acid, Free or Combined (Qualitative 
Test) [6) — Official Final Action 

(Applicable in presence of phosphates) 

Shake ca 5 g sample repeatedly with ca 250 mL cold H 2 in 
flask, and let insol. portion settle. Decant soln thru filter, and 
evap. filtrate to dryness. Powder residue, add few drops 1% 
resorcinol soln, 31.146, and ca 3 mL H 2 S0 4 , and heat slowly. 
Tartaric acid is indicated by rose-red, discharged on diln with 
H 2 0. 

Cream of Tartar and Free Tartaric Acid in 
Tartrate Powders (7) — Official Final Action 

Total, Combined, and Free Tartaric Acid 



8.014 



Determination 



To 2.5 g sample in 250 mL vol. flask add 100 mL H 2 at ca 50°, 
and hold at room temp, ca 30 min, shaking occasionally. Cool, 
dil. to vol. with H 2 0, shake vigorously, and filter thru large fluted 
paper. Pipet 2 portions of 100 mL each of clear filtrate into 250 
mL beakers, and evap. to ca 20 mL. To 1 portion add 3.5 mL ca 
1/V KOH. Mix well, and add 2 mL HOAc. Again mix well and add 
100 mL alcohol, stirring constantly. Treat other portion similarly, 
but use 1/V NaOH instead of KOH. Then treat each mixt. sep. as 
follows: Cool to ca 15°, stir vigorously ca 1 min, and leave in 
refrigerator overnight. Collect ppt in gooch on thin, tightly 
tamped pad of asbestos. Rinse beaker with ca 75 mL ice-cold 
80% alcohol, carefully washing down sides of beaker. Finally 
wash sides of crucible with 25 mL alcohol and suck dry. Transfer 
contents of crucible to original beaker with ca 100 mL hot H 2 0, 
and titr. with 0.1/V alkali, using phthln. Designate titer of portion 
treated with KOH as "x" and that treated with NaOH as "y." 



8.015 



Calculations 



% Total tartaric acid = 1.5(x + 0.6). 

% Cream of tartar = 1.88(y + 0.6). 

% Free tartaric acid = 1.5(x - y). 
In above formulas "0.6" represents solubility of cream of tartar 
in reaction mixt. in terms of 0.1/V alkali. 



Of Sodium Aluminum Phosphate (5) 
AOAC-Food Chemicals Codex Method 

8.011 Apparatus 

Magnetic stirrer-hot plate. — Must be capable of bringing assay 
soln to bp within 5 min. Alternatively, use ordinary hot plate 
and manual stirring. 



Free Tartaric Acid {Direct Determination) 



8.012 



Determination 



Accurately weigh ca 0.84 g sample, transfer to 250 mL beaker, 
and add 20 g NaCI, 5 mL 10% Na citrate.2H 2 soln, and 25 mL 
H 2 0. Pipet (or deliver from buret) 120 mL stdzd 0.1/V NaOH, 
50.032-50.034, swirling during addn. Stir on mag. stirrer-hot 
plate at slow to medium speed; avoid spattering. Bring to bp in 
3-5 min, and boil exactly 5 min. Remove from hot plate, and 
immediately cool to 25°. Titr. immediately with mag. stirring to 
pH 8.5 with stdzd 0.2/V HCI, 50.011-50.012, using pH meter 
previously stdzd with pH 7.0 buffer. Stir addnl 5 min and add 
0.2/V HCI to obtain pH 8.5. 

Neutzg value = (V,N, - V 2 N 2 ) x (0.84 x 10)/W, 
where V, and /V t = vol. and normality of NaOH, resp.; V 2 and N 2 
= vol. and normality of HCI, resp.; and W = g sample. 



8.016 



Reagent 



Saturated alcohol. — To ca 50 g finely powd pure cream of 
tartar in erlenmeyer add ca 100 mL alcohol and 100 mL H 2 0, 
shake vigorously several min, and let stand 15 min, shaking 
occasionally. Filter on paper in buchner; wash with ca 200 mL 
alcohol (1 + 1), then with alcohol, and finally with ether. Dry at 
temp, of boiling H 2 0. To 500 mL absolute alcohol add ca 5 g of 
the purified cream of tartar and let stand 2 hr, shaking occa- 
sionally. Properly purified cream of tartar requires ^0.15 mL 
0. 1/V alkali to neutze 100 mL of mixt. of 50 mL CHCI 3 and 150 mL 
of the satd alcohol. 



8.017 



Determination 



Weigh 1.25 g sample into absolutely dry 200 mL vol. flask, 
add 50 mL CHCI 3 , and let stand ca 5 min, shaking occasionally. 
(Discard detn if upon addn of CHCI 3 , powder sticks to bottom of 
flask, indicating moisture.) Add 100 mL satd alcohol, shake ca 
5 min, and let stand 30 min, shaking at frequent intervals. (It is 
not necessary to filter the alcohol reagent.) Dil. to vol. with the 
satd alcohol, shake few min, and filter thru large fluted paper. 
Titr. 100 mL clear filtrate with 0.1/V alkali, using phthln. Vol. (mL) 
alkali used x 1.2 = % free tartaric acid. 



AOAC Methods (1980) 



Aluminum 



145 



8.018 Free Tartaric Acid (Qualitative Test) 

Ext 5 g sample with absolute alcohol and evap. alcohol from 
ext. Dissolve residue in NH 4 OH (1 + 10), transfer to test tube, add 
good-size crystal of AgN0 3 , and heat gently. Tartaric acid is 
indicated by formation of Ag mirror. (If desired, ale. ext may be 
tested as in 8.013.) 



Starch — Official Final Action 

8.019 Direct Acid Hydrolysis Method 

(For baking powders and baking chemicals free from Ca) 

Stir 5 g sample 1 hr in 250 mL beaker with 50 mL cold H 2 0. 
Transfer to filter and wash with 250 mL cold H 2 0. Heat insol. 
residue 2.5 hr with 200 mL H 2 and 20 mL HCI (sp gr 1.125, 
52.002(c)) in flask provided with reflux condenser. Cool, and 
nearly neutze with NaOH. Transfer to 250 mL vol. flask, dil. to 
vol., filter, and det. glucose in aliquot of filtrate as in 31.038. Wt 
glucose obtained x 0.925 = wt starch. 

8.020 Indirect Method (8) 

(For baking powders and baking chemicals contg Ca) 

Mix 5 g sample with 200 mL HCI (1 + 11) in 500 mL vol. flask 
and let mixt. stand 1 hr, shaking frequently. Filter on 11 cm 
hardened paper, taking care to obtain clear filtrate. Rinse flask 
once without attempting to remove all starch, and wash paper 
twice with cold H 2 0. Carefully wash starch from paper back into 
flask with 200 mL H 2 0. Add 20 mLHCI (sp gr 1.125) and proceed 
as in 8.019. (Treatment with HCI, without dissolving starch, 
effectively removes Ca, which otherwise would be pptd as 
tartrate by alk. Cu soln.) 

Aluminum 
Qualitative Test (9) — Official Final Action 

(In presence of phosphates) 

8.021 Reagents 

(a) Ammonium acetate so/n. — 50%. Dissolve 50 g NH 4 OAc in 
50 mL H 2 0. 

(b) Aurintricarboxylic acid soln. — 0.1%. Dissolve 0.1 g aurin- 
tricarboxylic acid in H 2 and dil. to 100 mL. 



Table 8:01 Operating Parameters 



8.022 



Detection 



Place 1 g sample in 250 mL beaker, add 5 mL ca 1/V HCI and 
20 mL H 2 0, and heat until starch hydrolyzes. Add 100 mL cold 
H 2 0, 5 mL 70% NaNH A HP0 A .4H 2 soln, and 3 drops Me orange. 
Add NH 4 OH dropwise until ppt forms or color changes; then 
add 1/V HCI dropwise until ppt dissolves or color changes plus 
2 or 3 drops excess. Add 5 mL aurintricarboxylic acid soln and 
let stand 1 min. Add 50% NH 4 OAc soln dropwise until ppt forms 
or color changes and then 1 mL excess. Let stand 5 min, stirring 
occasionally, and filter. Bright red ppt on filter paper indicates 
presence of Al. 

Atomic Absorption Spectrophotometric 
Method (70)— Official First Action 

8.023 Apparatus 

Atomic absorption spectrophotometer. — Perkin-Elmer Model 
303, or equiv. Typical operating parameters for this app. are 
given in Table 8:01. Operator must become familiar with opti- 
mum settings for his own app. and use table only as guide. 
{Caution: See 51.006.) 



Wavelength, nm 

Slit width, mm 

Source, ma 

N 2 0, aspirating 

N 2 0, auxiliary and aspirating 

C 2 H 2 fuel 

Flame 

Ht, burner to light path, in. 

Sample uptake, mL/min 



309.3 

1 

30 

4.5 {scale divisions) 

5.5 (scale divisions) 

6 (metal ball scale division) 

reducing 

% 

4 



Optimum concn range, M-g/mL 50-150 



8.024 



Reagents 



(Do not use <2 mL pipets or <25 mL vol. flasks. Stock solns and 
(1 + 10) dilns may be stored indefinitely.) 

(a) Diluting soln.— To 500 mL H 2 add 20 mL H 2 S0 4 and 2.5 
g NaCI; dil. to 1 L with H 2 0. 

(b) Aluminum std solns.— {1) Stock soln—1 mg AI/mL. Dis- 
solve 1.000 g pure Al wire in min. amt HCI. Evap. almost to 
dryness, add 500 mL H 2 0, 20 mL H 2 S0 4 , and 2.5 g NaCI, and dil. 
to 1 L with H 2 0. (2) Working solns. — Dil. aliquots of stock soln 
with dilg soln, (a), to make s*4 std solns within concn range of 
instrument. 



8.025 



Preparation of Sample 



{Caution: See 51.019.) 

Accurately weigh ca 1 g sample into 250 mL Kjeldahl flask, 
add 2.0 mL H 2 S0 4 , and then slowly add 3 mL 30% H 2 2 . When 
initial vigorous reaction subsides, apply heat from Bunsen flame 
until sample begins to char. Add addnl 1 mL increments H 2 2 
and heat until soln no longer chars; finally heat to fumes of S0 3 . 
Cool, add 50 mL H 2 and 1 Pyrex glass chip, and boil 3-5 min. 
Cool and filter, if necessary, thru Whatman No. 2 paper into 100 
mL vol. flask, washing paper thoroly with H 2 0, and dil. to vol. 
Prep, reagent blank of 2.0 mL H 2 S0 4 and same total amt 30% 
H 2 2 used for sample. Measure A directly or dil. with dilg soln, 
(a), within range of instrument. 



8.026 



Determination 



Set up app. as in Table 8:01, or use previously established 
optimum settings for app. Zero app. while aspirating dilg soln, 
(a). Det. A of ^4 std solns within anal, range, alternating with 
sample soln readings. Flush burner with dilg soln, (a), and check 
point between readings. Correct for reagent blank reading if 
significant, and det. Al content from std curve of A against /u,g 
AI/mL: 

% Al - fag AI/mL) x {F/g sample) x 10"\ 
where F = 100 or 100 x mL final diln/mL aliquot, if original 100 
mL is dild. 

8.027 Insoluble Ash and Preparation of Solution (11) 
Official Final Action 

Char 5 g sample in Pt dish at heat below redness (ca 500°). 
Boil carbonaceous mass with HCI (1+2.5), filter into 500 mL vol. 
flask, and wash with hot H 2 0. Return residue, together with 
paper, to Pt dish, and burn to white ash. Boil again with the dil. 
HCI, filter, wash, combine filtrates, and dil. to 500 mL Incinerate 
residue after last filtration and weigh ash insol. in acid. 

8.028 Iron and Aluminum (77) — Official Final Action 

Sep. Si0 2 , if necessary, from 100 mL aliquot prepd soln, 8.027. 
Mix soln with excess 70% Na 2 HPO A .12H 2 soln. Add NH 4 0H 
until permanent ppt is obtained, then HCI dropwise until ppt 



146 



8. Baking Powders and Baking Chemicals 



AOAC Methods (1980) 



dissolves. Bring soln to bp and boil 2-3 min; mix with consid- 
erable excess 50% NH 4 0Ac soln, 8.021(a), and 4 mL HOAc (4+1). 
As soon as ppt of AIP0 4 , mixed with FeP0 4 , settles, collect on 
filter, wash with hot H 2 0, ignite, and weigh. Fuse mixed phos- 
phates with 10 parts Na 2 C0 3 , dissolve in H 2 S0 4 (1+6), reduce 
with Zn, and det Fe by titrn with std KMn0 4 soln (1 mL — 1 mg 
Fe). Det. P 2 5 in aliquot from 8.027 as in 8.033 or 7.119. 
Wt Al 2 3 = wt mixed phosphates - wt (Fe 2 3 + P 2 5 ). 

8.029 Calcium (77)— Official Final Action 

Heat combined filtrate and washings obtained in 8.028 to 50°, 
and add excess satd NH 4 oxalate soln. Let stand in warm place 
until ppt settles, filter, wash ppt with hot H 2 0, dry, and ignite 
over Bunsen burner and finally over blast lamp at ^950°. Cool 
in desiccator and weigh as CaO. 

8.030 Potassium and Sodium (77) — Official Final Action 

Evap. aliquot prepd soln, 8.027, nearly to dryness to remove 
excess HCI, dil., and heat to bp. While soln is still boiling add 
10% BaCI 2 .2H 2 soln as long as ppt forms, and then enough 
satd Ba{OH) 2 soln to make liq. strongly a Ik. After ppt settles, 
filter, and wash with hot H 2 0; heat filtrate to bp, add enough 
(NH 4 ) 2 C0 3 soln (1 part (NH 4 ) 2 C0 3 in 5 parts NH 4 OH soln (1 + 12)) 
to ppt all the Ba, filter, and wash with hot H 2 0. Evap. filtrate to 
dryness and ignite residue below redness to remove NH 4 salts. 
Add little H 2 and few drops (NH 4 ) 2 C0 3 soln to residue. Filter 
into weighed Pt dish, evap., ignite below redness, and weigh 
mixed K and Na chlorides. 

Digest residue with hot H 2 0, filter thru small filter, and dil. 
filtrate, if necessary, to provide s*20 mL liq. for each 100 mg 
K 2 0. Acidify with few drops HCI and add excess Pt soln (10.5 g 
H 2 PtCI 6 /100 mL). Evap. on H 2 bath to thick paste; treat residue 
repeatedly with 80% alcohol, decanting thru weighed gooch or 
other filter; transfer ppt to filter, and wash thoroly with 80% 
alcohol. Dry 30 min at 100° and weigh. Calc. K found to its equiv. 
of KCI and subtract result from wt mixed chlorides to obtain wt 
NaCI. 



Phosphorus — Official Final Action 



8.031 



Reagents 



(a) Ammonium nitrate soln. — Dissolve 100 g P-free NH 4 N0 3 
in H 2 and dil. to 1 L. 

(b) Magnesia mixture. — (7) Dissolve 55 g cry std MgCI 2 .6H 2 
in H 2 0, add 140 g NH 4 CI and 130.5 mL NH 4 OH, and dil. to 1 L 
Or, (2) dissolve 55 g crystd MgCI 2 .6H 2 in H 2 0, add 140 g NH 4 CI, 
dil. to 870 mL, and add NH 4 OH to each required portion of soln 
just before using, at rate of 15 mL/100 mL soln. 

(c) Ammonium hydroxide soln for washing. — (1+9). Should 
contain ^2.5% NH 3 by wt. 

8.032 Preparation of Solution 

Mix 5 g sample with little Mg(N0 3 )2 soln, 2.019, dry, ignite, 
dissolve in HCI (1+2.5), and dil. to definite vol. In aliquot of soln 
det. P as in 8.033 or 7.119. 



8.033 



Determination 



Pipet aliquot of prepd soln into 250 mL beaker; add NH 4 OH 
in slight excess and barely dissolve ppt formed with few drops 
HN0 3 , stirring vigorously. If HCI or H 2 S0 4 has been used as solv., 
add ca 15 g cryst. NH 4 N0 3 or soln contg that amt. To hot soln 
add 70 mL molybdate soln, 7.118(a), for every 100 mg P 2 5 
present. Digest 1 hr at ca 65° and test for complete pptn of P 2 5 



by adding more molybdate soln to clear supernate. Filter, and 
wash with cold H 2 or preferably with the NH 4 N0 3 soln. Dissolve 
ppt on filter with NH 4 OH (1+1) and hot H 2 0, and wash into 
beaker to vol. =sl00 mL. Neutze with HCI, using litmus paper or 
bromothymol blue as indicator; cool, and from buret slowly add 
(ca 1 drop/sec), stirring vigorously, 15 mL magnesia mixt./100 
mg P 2 5 present. After 15 min add 12 mL NH 4 OH and let stand 
until supernate is clear (usually 2 hr); filter, wash ppt with 
NH 4 OH (1+9) until washings are practically Cl-free, dry, burn at 
low heat, and ignite to const wt, preferably in furnace at 
950-1000°; cool in desiccator, and weigh as Mg 2 P 2 7 . Report as 
% P 2 5 . 

8.034 Qualitative Test — Official Final Action 

Add 10 mL H 2 to 1-2 g sample in 150 mL beaker. Make just 
acid with HN0 3 , filter, take equal vols filtrate and molybdate 
soln, 7.1 18(a), and warm at 40-50°. Yellow ppt indicates presence 
of phosphate. 

8.035 Sulfate (72)— Official Final Action 

Boil 5 g sample 1.5 hr with mixt. of 300 mL H 2 and 15 mL 
HCI. Filter, wash filter thoroly with hot H 2 0, cool combined 
filtrate and washings, and dil. to 500 mL wjth H 2 0. Det. sulfate 
in 100 mL aliquot as in 3.062. 

8.036 Ammonia — Official Final Action 

To 2 g sample in distn flask add 300-400 mL H 2 and excess 
of NaOH soln (1 + 1), connect with condenser, and distil into 
measured vol. std acid. Titr. excess acid in distillate with std 
alkali, using Me red. 

8.037 Arsenic — Official Final Action 

Place 5 g sample directly in generator, 25.007(a); add 10 mL 
H 2 0, little at time to prevent foaming over, and then 15 mL As- 
free HCI, adding it dropwise until foaming ceases. Heat on steam 
bath until drop of mixt., when dild and treated with I soln, does 
not show blue. Then dil. to ca 30 mL with H 2 and continue as 
in 25.010 or 25.012, beginning with addn of Kl reagent. Prep, 
blank and stds for comparison, using As-free HCI of same concn 
as that used in detn. 

8.038 Fluorine — Official Final Action 
See 25.049-25.055. 

8.039 Lead— Official Final Action 

See 25.061-25.062 and 25.095-25.105. 



SELECTED REFERENCES 

(7) JAOAC 6, 453(1923). 

(2) JAOAC 10, 36(1927). 

[3) JAOAC 31, 278(1948); 32, 83, 269(1949); 33, 77(1950). 
[4] JAOAC 33, 77(1950); 34, 296(1951). 

(5) JAOAC 59, 26(1976). 

(6) Ann. chim. anal. 4, 263(1899). 

(7) JAOAC 13, 385(1930); 22, 599(1939). 

{8) Conn. Agr. Expt. Sta. Rpt. 1900 (II), p. 174. 
{9) J. Am. Chem. Soc. 47, 142(1925); JAOAC 34, 61(1951); 35, 
57(1952). 
(10) JAOAC 55, 684(1972). 
(7 7) Conn. Agr. Expt. Sta. Rpt. 1900, p. 178. 
(72) USDA Bur. Chem. Bull. 13 (V), p. 596; Conn. Agr. Exp. Sta. 
Rpt. 1900, p. 179. 



9, Beverages: Distilled Liquors 



SPIRITS 

9.001 Physical Examination — Procedure 

Note and record following: (a) Color and depth of color; (b) 
odor— whisky, brandy, rum, etc., or foreign; (c) taste — whisky, 
brandy, rum, etc., or foreign. 



9.007 



Determination 



Color (7) — Official Final Action 



9.002 



Definition 



Whisky color units are defined as 10 x A at 430 nm, measured 
in monochromatic light, of sample Y 2 " thick which has spectral 
color characteristics of an av. whisky free of turbidity. 

This definition applies only to A values obtained with precise 
spectrophtr with band width of ^1 nm at 430 nm, and whose 
wavelength and photometer scales have been checked and 
corrected by methods recommended by NBS, in LC-1017, Jan. 
1967, and in SP260-^1. 



Potassium Bichromate Calibration Method 
9.003 Preparation of Standard Curve 

Prep, solns of K 2 Cr 2 7 in 0.01/V H 2 S0 4 as follows: 



Color 




Color 




Unit 


g/L 


Unit 


g/L 


1 


0.0500 


6 


0.3000 


2 


0.1000 


7 


0.3500 


3 


0.1500 


8 


0.4000 


4 


0.2000 


9 


0.4500 


5 


0.2500 


10 


0.5000 



Read A of these solns in spectrophtr at 430 nm against H 2 0, 
using same size cell as used in detns. If other than !4" cell is 
used, convert reading to this size. Plot color units against A or 
calc. av. factor for converting instrument reading to color units 
if straight line is obtained. 



9.004 



Determination 



Place sample, or sample dild with 50% alcohol, in cell and 
det. A against H 2 0. Calc. color units, using factor or std curve. 



Pipet 20 mL whisky into cylinder, 9.005(b). Add by pipet, in 
order, 10 mL satd NaCl soln, 0.5 mL HCI, and 10 mL Me Pr 
ketone. Immediately invert 10-1 5 times and let layers sep. Color 
in lower layer indicates presence of caramel, vegetable extrac- 
tives, or synthetic dye. Read vol. of org. layer within 1 hr and 
det. its ^ at 430 nm. If A is too great or if solv. layer is cloudy, 
dil. aliquot to known vol. with either 50% MeOH or 50% alcohol 
and read A. 



9.008 



Calculation 



Example: If from 20 mL sample, org. layer of 16.1 mL was 
obtained which had A of 0.420 in 1 cm cell after dil n 14-1 with 
50% alcohol: {16.1 x 0.420 x 12.7 x 2)/20 - 8.59 color units 
(Lovibond number), where 12.7 is conversion factor to color 
units. 

Specific Gravity (Apparent) — Official Final Action 
9.009 Apparatus 

(a) Constant temperature water bath. 

(b) Pycnometers. —100 and 50 mL (Fig. 9:01). 



9.010 



Calibration 



Fill thoroly cleaned pycnometer with recently distd H 2 0, 
stopper, and immerse in const temp. H 2 bath with bath level 
above graduation mark on pycnometer. After 30 min, remove 
stopper and with capillary tube adjust until bottom of meniscus 
is tangent to graduation mark. With small roll of filter paper, dry 
inside neck of pycnometer, stopper, and immerse in H 2 at 
room temp. 15 min. Remove pycnometer, dry, let stand 15 min, 
and weigh. Empty pycnometer, rinse with acetone, and dry 
thoroly in air with suction. Let empty flask come to room temp., 
stopper, and weigh. Wt in air of contained H 2 - wt filled 
pycnometer - wt empty pycnometer. 

9.011 Determination 

Obtain wt sample as in 9.010. 

Sp gr in air = S/W, where S - wt sample, and W = wt H 2 0. 



Natural and Artificial Coloring Matter (Organic and 
Water-Soluble Color) (2)— Official Final Action 

Spectrophotometric Method 

9.005 Apparatus 

(a) Spectrophotometer. — See 9.002. 

(b) Graduated cylinder. — Cylindrical type of uniform diam., 
with pressed or molded base and? stopper. Distance from base 
to top is 285-295 mm. To contain 50 mL at 20°, graduated in 0.2 
mL with each fifth mark distinguished by longer line; numbered 
from bottom upward at 2 mL intervals; error of graduations 
=£0.2 mL at any point. {Available from SGA Scientific Inc., No. 
JC-9675.) 

9.006 Reagents 

(a) n-Methyl propyl ketone. — 2-Pentanone, practical. 

(b) Saturated sodium chloride soln.— Use USP or ACS NaCl. 

(c) Alcohol. — MeOH, reagent grade, or alcohol, USP. 



Alcohol by Volume 

From Specific Gravity by Pycnometer [3) 
Official Final Action 

9.012 Apparatus 

Distillation apparatus. — 500 mL flask, connected thru bulb 
(Iowa State type is convenient) to vertically assembled Liebig 
condenser with jacket s=400 mm long, inner tube 9±1 mm id, 
with adapter. Joints may be live rubber orf . Heat with elec- or 
gas-operated unit. 



9.013 



Determination 



(Some samples do not require distn prior to detn, e.g., straight 
bourbon whisky; alcohol-H 2 mixts contg traces of volatile 
ingredients.) 

(a) Samples containing 60% or less alcohol by volume. — 
Calibrate 100 mL pycnometer, Fig. 9:01, as in 9.010, at one of 



147 



148 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



Ground in 
Stopper 






s 




2 

c 
o 

CO 

- 3 

CO 

Ik. 

U 



EE 
EE 



no. 
contains 
100 ML 
20° C 



|-*~35mm — *-] 



58mm 




11mm 
OD 



\ 



E 

E 






Q 
O 






N 



e 

E 

OS 




no. 

contains 

50 ML 

20° C 



-34mm 
.48mm 



FIG. 9:01 — 100 mL and 50 mL pycnometers 



temps specified in 52.003. Fill clean, dry pycnometer with sample 
and adjust to vol. at calibration temp, as in 9.010. 

Transfer contents of pycnometer to distg flask, just previously 
rinsed with cold H 2 and contg few glass beads, or equiv. Rinse 
pycnometer 3 times, using total of 25 mL cold H 2 (40 mL for 
cordials or wines), and add rinse H 2 to flask. Place wet 
pycnometer so that adapter extends just into bulb. Surround 
pycnometer with ice or ice-H 2 0. Complete connections and pass 
thru condenser rapid stream of H 2 kept at ^25° at outlet. Distil 
ca 96 mL at uniform rate in 5=30 but ^60 min, using longer 
times for higher percentages of alcohol. Remove and stopper 
pycnometer, mix distillate by swirling, and wash down with H 2 
any drops that may be above graduation mark. Immerse in 
const temp, bath at calibration temp, and after 30 min carefully 
dil. to vol., with aid of capillary tube, by adding H 2 previously 
boiled and cooled to same temp. Det. sp gr of distillate as in 
9.011. Obtain corresponding % alcohol by vol. from 52.003. 
(This result is % alcohol by vol. at 15.56° (60°F).) 

(b) Samples containing more than 60% alcohol by volume. — 
Proceed as in (a) with following changes: Calibrate 100 mL and 
50 mL pycnometers, Fig. 9:01, at 15.56°, fill 50 mL pycnometer 
with sample, and adjust to vol. at 15.56°. Add 50 mL cold H 2 
to distg flask before transfer of sample and collect distillate in 
100 mL pycnometer. Adjust to vol. at 15.56°. Obtain sp gr of 
distillate, and from table, 52.003, obtain % alcohol by vol. in 
distillate. Calc. as follows: 

% alcohol by vol. in sample at 15.56° = D x W/W; 
where D = % alcohol by vol. in distillate at 15.56°; W = wt H 2 
at 15.56° in 100 mL pycnometer; and W = wt H 2 at 15.56° in 
50 mL pycnometer. 



From Specific Gravity by Hydrometer [4) 
Official Final Action 

(Applicable to spirits contg ^600 mg ext/100 mL) 

9.014 Apparatus 

(a) Hydrometer. — Graduated to 0.1 or 0.2° proof, with calibra- 
tion corrections. 

(b) Thermometer.— Graduated to 0.25 or 0.5°F, with calibra- 
tion corrections. 

(c) Cylinder. — Clear glass, 2.5" diam., 14" high. 

(d) Metal clips. — To hold thermometer in cylinder. 



9.015 



Determination 



Clean and dry hydrometer before use. Let hydrometer, ther- 
mometer, cylinder, and sample come to room temp. Rinse 
cylinder, contg thermometer held in place by spring frame clip, 
2 or 3 times with portion of sample. Fill cylinder to desired level 
with sample, holding cylinder at ca 45° angle to reduce agitation 
and air bubbles. (After hydrometer is inserted, liq. level should 
be slightly below rim of cylinder.) Place palm of hand over top 
of cylinder and slowly invert 3 or 4 times to equalize temps of 
liq. and cylinder. Wipe off any liq. on outside of cylinder. (Do 
not place hands on cylinder in such way as to warm liq. inside.) 
Insert hydrometer in liq.; then raise and lower hydrometer bulb 
from top to bottom 5 or 6 times to temper and distribute slight 
temp, changes thruout liq. Keep hydrometer bulb in liq., dry 
stem, and let hydrometer come to rest without wetting more 
than few tenths degrees of exposed stem. 

Read hydrometer, then thermometer. To read hydrometer 



AOAC Methods (1980) 



Alcohol 



149 



scale, place eye slightly below plane of surface of liq., and then 
slowly raise head, keeping eye perpendicular to hydrometer, 
until surface flattens from ellipse into straight line. Take point 
where this line intersects hydrometer scale as reading of hy- 
drometer. 

Raise hydrometer slightly above its point of rest and again let 
it come to rest in liq. Read hydrometer and thermometer again 
to verify original readings. Read hydrometer to nearest 0.02° 
and thermometer to nearest 0.1°. Remove and dry hydrometer. 
Reinvert cylinder and contents several times (with thermometer 
left in place) to thermally equilibrate system. Retemper hydrom- 
eter, dry stem, and again read hydrometer and thermometer. 
Apply calibration corrections for both hydrometer and ther- 
mometer. Calc. true % of proof from Table No. 1 of the U.S. 
Treasury Department Gauging Manual, 1970. Average calcd 
values if they agree within 0.1° proof; otherwise take addnl 
readings and average. 

Det. ext as in 9.022, and for every 100 mg ext/100 mL add 0.4° 
proof to apparent proof. 

9.016 From Refraction — Official Final Action 

Measure 25 mL sample into distn flask, noting temp.; dil. with 
100 mL H 2 0, distil nearly 100 mL, dil. to vol. at same temp., and 
det. immersion refractometer reading. Obtain corresponding 
% alcohol from 52.004. 

When vol. measurements are made at temp, other than 1 5.56°, 
multiply % alcohol from 52.004 by appropriate factor from Table 
9:01. 



/ 




j 
I5ml 


7. 


7.5ml 


10 
20 
25 
30 
35 
40 
45 

50 

55 

SO 


|t 



FIG. 9:02— Williams tube 



Williams Field Test (5) — Procedure 



9.017 



Apparatus 



Williams tube.— See Fig. 9:02. Available from Kontes Glass 
Co., No. K-899400. Clean frequently and dry. 

9.018 Reagents 

(a) Dilute hydrochloric ac/cr.—Dil. 10 mL HCI to 100 mL with 
H 2 0. 

(b) Solvent. — Mix 70 mL Pentasol or isoamyl alcohol, 28 mL 
toluene, and 2 mL dil. HCI. Shake well until acid completely 
dissolves. 



times to mix intimately. Stand tube upright and let sep. When 
sepn is complete, rotate tube to shake down globules of lower 
soln that adhere to sides, and stopper. When settling and 
drainage are complete, read % alcohol (by vol.) where meniscus 
between the 2 layers falls on calibration mark. Repeat mixing 
and settling, and read again. 

9.020 Temperature Correction 

Correct for temp, and alcohol concn according to Table 9:02. 

Example: % alcohol from tube: 48.0% at 90°F. Correction 
factor from table: -1.1; 48.0 - 1.1 = 46.9%. 



9.019 



Determination 



Place sample in tube, accurately adjusting bottom of meniscus 
to coincide with 7.5 mL mark. Remove any excess sample on 
sides of tube above 7.5 mL mark with swab or roll of filter paper. 
Add solv. to 15 mL mark. Stopper tube and invert number of 



9.021 Alcohol by Weight— Official Final Action 

Accurately weigh 40-50 g sample in clean, dry 50 mL pyc- 
nometer, Fig. 9:01, or other closed vessel. (If alcohol is ^60% 
by vol., the 100 mL sample of 9.013(a) may be weighed and 
used,) Transfer to 500 mL distn flask contg 50 mL H 2 and few 



Table 9:01 


Factors for calculating original alcohol content for 25 and 50 mL samples distilled to 100 mL when refractometer 






measurements are made at 20, 


25, 


30, 


or 35° 












25 mL Sample 










50 mL Sample 








Temp, of Measurement 






Temp, of Measurement 




Ale. % by Vol 


20° 


25° 


30° 


35° 


20° 


25° 


30° 


35° 


in Distillate 






















at 15.56° 




Multiply Alcohol 


in Distillate by: 






Multiply Alcohol 


in Distillate by: 




0-3.99 


4.001 


4.002 


4.003 


4.004 






2.000 


2.000 


2.000 


2.000 


4-5.99 


4.003 


4.006 


4.009 


4.013 






2.000 


2.001 


2.001 


2.001 


6-7.99 


4.005 


4.011 


4.016 


4.021 






2.001 


2.001 


2.002 


2.002 


8-9.99 


4.007 


4.015 


4.023 


4.030 






2.001 


2.002 


2.003 


2.004 


10-11.99 


4.009 


4.019 


4.028 


4.037 






2.001 


2.003 


2.004 


2.006 


12-13.99 


4.010 


4.021 


4.031 


4.041 






2.002 


2.004 


2.006 


2.007 


14-15.99 


4.011 


4.022 


4.032 


4.043 






2.002 


2.005 


2.007 


2.009 


16-19.99 


4.011 


4.023 


4.034 


4.045 






2.003 


2.006 


2.008 


2.011 


20-more 


4.011 


4.024 


4.036 


4.047 






2.003 


2.006 


2.009 


2.012 



150 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



Table 9:02. Temperature (°F) correction factors 



% 


60° 


62° 


64° 


66° 


63° 


70° 


72° 


74° 


76° 


78° 


43 


+ 0.5 


+ 0.3 


+ 0.2 


0.0 


-0.1 


-0.2 


-0.4 


-0.5 


-0.7 


-0.8 


43.4 


+ 0.5 


+ 0.4 


+ 0.2 


+0.1 


0.0 


-0.2 


-0.3 


-0.5 


-0.6 


-0.7 


44 


+ 0.6 


+ 0.4 


+ 0.3 


+0,2 


0.0 


-0.1 


-0.3 


-0.4 


-0.5 


-0.7 


45 


+0.7 


+ 0.5 


+ 0.4 


+0.3 


+0.1 


0.0 


-0.2 


-0.3 


-0.4 


-0.6 


46 


+ 0.8 


+0.7 


+ 0.5 


+ 0.4 


+ 0.2 


+ 0.1 


0.0 


-0.2 


-0.3 


-0.5 


47 


+ 0.9 


+ 0.8 


+ 0.6 


+0.5 


+0.3 


+0.2 


+ 0.1 


-0.1 


-0.2 


-0.4 


47.5 


+ 1.0 


+ 0.8 


+ 0.7 


+ 0.5 


+0.4 


+ 0.3 


+ 0.1 


0.0 


-0.2 


-0.3 


48 


+ 1.1 


+ 0.9 


+ 0.7 


+ 0.6 


+ 0.4 


+ 0.3 


+ 0.2 


0.0 


-0.1 


-0.2 


49 


+ 1.1 


+ 1.0 


+ 0.8 


+ 0.7 


+0.6 


+0.4 


+ 0.3 


+0.1 


0.0 


-0.1 


50 


+ 1.2 


+ 1.1 


+0.9 


+ 0.8 


+0.7 


+ 0.5 


+ 0.4 


+0.3 


+0.1 


0.0 


% 


80° 


82° 


84° 


86° 


8B° 


90° 


92° 


94° 


96° 


98° 


43 


-0.9 


-1.1 


-1.2 


-1.4 


-1.5 


-1.6 


-1.8 


-1.9 


-2.0 


-2.2 


43.4 


-0.9 


-1.0 


-1.2 


-1.3 


-1.4 


-1.6 


-1.7 


-1.9 


-2.0 


-2.2 


44 


-0.8 


-0.9 


-1.1 


-1.2 


-1.4 


-1.5 


-1.6 


-1.8 


-1.9 


-2.1 


45 


-0.7 


-0.8 


-1.0 


-1.1 


-1.3 


-1.4 


-1.5 


-1.7 


-1.8 


-2.0 


46 


-0.6 


-0.7 


-0.9 


-1.0 


-1.2 


-1.3 


-1.4 


-1.6 


-1.7 


-1.8 


47 


-0.5 


-0.6 


-0.8 


-0.9 


-1.0 


-1.2 


-1.3 


-1.5 


-1,6 


-1.7 


47.5 


-0.4 


-0.6 


-0.7 


-0.8 


-1.0 


-1.1 


-1.3 


-1.4 


-1.5 


-1.7 


48 


-0.4 


-0.5 


-0.6 


-0.8 


-0.9 


-1.1 


-1.2 


-1.3 


-1.5 


-1.6 


49 


-0.3 


-0.4 


-0.5 


-0.7 


-0.8 


-1.0 


-1.1 


-1.2 


-1.4 


-1.5 


50 


-0.2 


-0.3 


-0.4 


-0.6 


-0.7 


-0.9 


-1.0 


-1.1 


-1.3 


-1.4 



clean glass beads, or equiv. Rinse pycnometer 3 times, bringing 
contents of distn flask to ca 125 ml_. Distil, and det. % alcohol 
by vol. in distillate as in 9.013(a). Det. corresponding % alcohol 
by wt in distillate from table, 52.005. Multiply result by wt 
distillate and divide by wt sample. 

9.022 Extract — Official Final Action 

Weigh, or measure at 20°, 25-100 mL sample, evap, to dryness 
on steam bath, dry 30 min at 100°, cool in desiccator 30 min, 
and weigh. 

9.023 Ash— Official Final Action 

Proceed as in 31.012 or 31.013, using residue from 9.022. 



Potassium 
Flame Photometric Method [6)— Official Final Action 

9.024 Reagent and Apparatus 

(a) Std so/n$.— Prep, as in 11.024(a), except make final dilns, 
i.e., 1-10 ppm K, with 50% alcohol. 

(b) Flame spectrophotometer. — See 11.024(b). 

9.025 Determination 

[Caution: See 51.007.) 

Proceed as in 11.025, except burn sample undild, or, if 
necessary, dild with 50% alcohol (usually 2.5-5.0 times). Calc. 
ppm K as in 11.025. 



Sodium 

Flame Photometric Method (6) — Official Final Action 

9.026 Reagent and Apparatus 

(a) Std so/rts.—Prep. as in 11.026, except make final dilns, 
i.e., 1-10 ppm Na, with 50% alcohol. 

(b) Flame spectrophotometer. — See 11.024(b). 



9.027 



Determination 



[Caution: See 51.007.! 



Proceed as in 11.025, except burn sample undild, or, if 
necessary, dild with 50% alcohol until %T falls within %T range 
of stds. Calc. ppm Na as in 11.025. 

9.028 Phosphorus — Official Final Action 
See 11.034. 



Copper — Official Final Action 
Atomic Absorption Method (7) 



9.029 



Reagent 



(Distil H 2 and alcohol from all-Pyrex stills 
into Cu-free receiver.) 



Copper std soins. — [l)Stocksoln. — 0.2 mg/mL Dissolve 0.393 
g CuS0 4 .5H 2 (free from any whitish deposit) in 500 mL vol. 
flask contg H 2 and 2 mL H 2 S0 4 . Dil. to vol. and mix. [2) Working 
soln. — 0.004 mg/mL. Prep, daily by dilg 2.00 mL stock soln to 
100 mL 

9.030 Preparation of Standard Curve 

To series of 50 mL vol. flasks each contg 25 mL alcohol, add 
0, 2, 4, 6, 10, and 12 mL Cu working std soln. Dil. nearly to mark 
with H 2 0, mix, and cool to room temp, before dilg to vol. Stds 
contain 0.0, 0.16, 0.32, 0.48, 0.80, and 0.96 ppm Cu (/-tg/mL), 
res p. 



9.031 



Determination 



[Caution: See 51.006.) 



Follow operating instructions supplied by manufacturer or 
previously established optimum settings for AA spectrophtr 
used. Adjust instrument to 0A while aspirating blank. Read at 
324.7 nm 5=4 std solns within anal, range before and after each 
6-10 samples. Prep, calibration curve from av, of each std before 
and after sample group. Use std curve to convert A values for 
samples to ppm Cu. 



AOAC Methods (1980) 



Iron 



151 



ZDBT Colorimetric Method (8) 

9.032 Reagents 

Prep. H 2 0, alcohol, and Cu stds as in 9.029, and in addn: 
Zinc dibenzyldithiocarbamate (ZDBT)-carbon tetrachloride 

soln.— 0.2%. Dissolve 2 g ZDBT (Uniroyal Chemical or ICN- 

K&K Laboratories) in 1 L CCI 4 by warming in H 2 bath at <77°. 

Filter thru Whatman No. 41, or equiv. acid-washed paper, into 

dark bottle. Store in refrigerator. 

9.033 Apparatus 

Separators. — 60 or 125 mL pear-shaped separators with Teflon 
stopcocks. Clean separators with hot chromic acid cleaning soln 
and rinse with H 2 0. Before each analysis, shake mixt. of 10 mL 
H 2 0, 0.5 mL 6/V H 2 S0 4 , and 10 mL ZDBT-CCI 4 soln in each 
separator 1 min. Clean inside of stems with cotton swab soaked 
in ZDBT-CCI4 soln. Drain and rinse separators with H 2 0. 

9.034 Preparation of Standard Curves 

(a) 80-135° proof alcoholic samples. — To separators contg 
5 mL alcohol, add 0, 0.50, 1.00, 2.00, and 3.00 mL Cu working 
std soln and 5, 4.5, 4, 3, and 2 mL H 2 0, resp. Solns in separators 
contain 0.0, 0.20, 0.40, 0.80, and 1.20 fxg Cu/mL (ppm), resp. 
Treat as in 9.035. Plot ppm Cu against A. 

(b) Aqueous samples, wines, and other low proof samples. — 
Prep, as in (a) except use 5 mL H 2 instead of alcohol. 



9.035 



Determination 



To separator contg 10 mL sample (dil. sample >135° proof to 
80-135° proof) or std, add 0.5 mL 6/V H 2 S0 4 and 10.0 mL ZDBT- 
CCI 4 soln. Stopper and shake briefly; release pressure by re- 
moving stopper. Replace stopper and shake vigorously 100 
times. If funnel stems are not dry, remove drops of liq. with 
small roll acid-washed paper (e.g., Whatman 41) to prevent 
draining H 2 drops into cell. Insert plug of fibre glass (Corning 
Glass Works No. 3950) or cotton into each stem to filter out 
possible haze materials. Within 10-60 min, det. A of CCI 4 layer 
at 438 nm. Let few mL CCI 4 layer pass thru filtering medium 
before collecting sample in cell. Use CCI 4 layer from appropriate 
ppm Cu soln (prepd as for std curve (a) or (b)) as ref. Det. Cu 
concn from appropriate std curve. Multiply by diln factor if 
sample was dild. 



Iron (5)— Official Final Action 
Atomic Absorption Method 

9.036 Apparatus 

Spectrophotometer. — Perkin-Elmer Corp. 303 (or later double 
beam model), or equiv., with 3-slot Boling burner head, or equiv. 

9.037 Reagent 

(Use Fe-free H 2 and reagents; rinse glassware with 
HCI and H 2 before use.) 

Iron std solns.— (1) Stock soln.— 10 /jug/ml. Dissolve 0.0684 g 
ferrous ethylenediammonium sulfate (FeC 2 H 4 (NH 3 ) 2 S0 4 .4H 2 0, G. 
Frederick Smith Chemical Co.) in H 2 0, add 2.5 mL H 2 S0 4 , dil. to 
1 L with H 2 0, and mix thoroly. (2) Working solns.— 0.0, 0.1, 0.2, 
0.3, and 0.4 ppm. To 100 mL vol. flask contg 50 mL 43% alcohol, 
add 0, 1, 2, 3, and 4 mL stock soln and dil. each soln nearly to 
vol. with 43% alcohol. Mix thoroly, let cool to room temp., and 
adjust to 100 mL with 43% alcohol. 



9.038 Determination 

(Caution: See 51.006.) 

Follow manufacturer's operating instructions, using lean air- 
C 2 H 2 flame and single element Fe lamp. Set wavelength at 248.3 
nm and adjust spectrophtr to A while aspirating blank (0.0 
ppm Fe). Analyze stds before and after duplicate series of 
samples. Det. av. A values. Aspirate H 2 between each detn to 
flush burner, and resets to with blank. Plot std curve of A 
against ppm Fe. Read ppm Fe in sample from this curve. 



TPTZ Colorimetric Method 

(Not applicable to brandy) 

9.039 Apparatus 

(a) Spectrophotometer. — Beckman Instruments Model DU, or 
equiv., or photoelec. colorimeter with suitable filter, e.g., Klett 
colorimeter with No. 60 filter, 

(b) Water bath. — To maintain const temp, at ca 60°. 



9.040 



Reagents 



(Use Fe-free H 2 and reagents; rinse glassware 
with HCI and H 2 before use.) 



(a) Hydroxyiamine hydrochloride soln. — 40%. Dissolve 10 g 
NH 2 OH.HCI in 25 mL H 2 0. 

(b) Ammonium perchlorate soln . — 1 0% . Dissolve 1 g 
NH 4 CI0 4 in 100mLH 2 O. 

(c) 1,2-Propanediol cyclic carbonate [propylene carbonate). — 
Reagent grade (MC/B Manufacturing Chemists). 

(d) 2,4,6-Tripyridyl-s-triazine (TPTZ) soln .—0.00 1/W. Add 
0.0781 g TPTZ (G. Frederick Smith Chemical Co.) to 100 mL H 2 
contg 5 drops HCI in 250 mL vol. flask. Dil. to vol. with H 2 and 
filter thru coarse fritted glass funnel. Store in Fe-free glassware. 

(e) Sodium acetate soln . — 40%. Dissolve 48.2 g anhyd. NaOAc 
in 75.7 mL H 2 and 24.3 mL HCI. Add 5 mL 0.001M TPTZ, 5 mL 
40% NH 2 OH.HCI, 2 mL 10% NH 4 CI0 4 , and 10 mL propylene 
carbonate. Add 5 mL CHCI 3 , shake, and discard lower layer. After 
2 CHCI3 extns, add 5 mL 0.001/W TPTZ and repeat CHCI 3 extn. 
(Four extns are necessary to obtain colorless soln.) Store in Fe- 
free glassware. 

(f) Ascorbic acid solns. — (7)5%. — Dissolve 2.5 g ascorbic acid 
in 25 mL H 2 and dil. to 50 mL with alcohol. Prep, fresh daily. 
(2) 5% Ascorbic acid in sodium acetate buffer. — Dissolve 2.5 g 
ascorbic acid in 20 mL H 2 0, add 5 mL 40% NaOAc, and dil. to 
50 mL with alcohol. Prep, fresh daily. 

(g) Iron std solns. — Prep, same concns as in 9.037, but prep. 
200 mL each working soln. 

9.041 Preparation of Standard Curve 

Pipet 50 mL43% alcohol contg known amts Fe (i.e., (reagent 
color blank), 0.1, 0.2, 0.3, and 0.4 ppm) into 5 sep. 100 mL vol. 
flasks. Add 2 mL 5% ascorbic acid in NaOAc buffer and 2 mL 
TPTZ, and heat 15 min in ca 60° H 2 bath. 

Cool solns to room temp, and det./4 on spectrophtr at 593 nm 
or photoelec. colorimeter with appropriate filter. Use 43% 
alcohol to zero colorimeter. Subtracts of reagent color blank 
from A of samples. Plot corrected A readings against ppm Fe 
to obtain std curve. (Straight line should be obtained with Fe 
concns used.) 



9.042 



Determination 



Pipet 50 mL sample into each of 2 sep. 100 mL vol. flasks and 
add 2 mL 5% ascorbic acid to each. Add 2 mL TPTZ to one flask 



152 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



and 2 mL H 2 to other (product blank). Heat 15 min in ca 60° 
H 2 bath. Cool solns and measure A of each soln as for std 
curve. Subtracts of reagent color blank, 9.041, and also A of 
product color blank from A of samples. Det. Fe concn from std 
curve. 



9.050 Preparation of Sample 

To 200 mL sample in 500 mL erlenmeyer, add ca 35 mL H 2 
and few grains SiC (Carborundum). Distil slowly into 200 mL 
vol. flask until distillate is nearly at mark. Dil. to vol. and mix. 



9.051 



Determination of Esters [11) 



Chloride {10)— Official Final Action 
9.043 Apparatus 

See 11.029. 



9.044 



Reagents 



See 11.030 and in addn: 

(a) Dilute chloride std soln. — Dil. 50 mL chloride std soln, 
11.030(c), to 500 mL with H 2 0. Alternatively, dissolve 0.2103 g 
KCI in H 2 and dil. to 1 L 1 mL = 0.1 mg CI. 

(b) Dilute silver nitrate std soln.— Dil. 50 mL std soln, 11.030(d), 
to 500 mL with H 2 0. Alternatively, dissolve 0.4791 g AgN0 3 in 
H 2 and dil. to 1 L 1 mL = 0.1 mg CI. 

(c) Alcohol soln.— Place 500 mL alcohol in 1 L vol. flask, add 
ca 475 mL H 2 0, mix, cool to room temp., and dil. to 1 L with 
H 2 0. 



9.045 



Determination 



Det. equivalence point voltage as in 11.031 except use alcohol 
soln instead of H 2 to adjust vol. to 100 mL. Use std solns 
11.030(c) and (d). 

Pipet 5 mL dil. std CI soln into 250 mL beaker, and add 95 mL 
alcohol soln and 1.0 mL HN0 3 . Titr. with dil. std AgN0 3 soln to 
predetd equivalence voltage as in 11.031. 

Pipet 100 mL distd spirits sample into 250 mL beaker, add 1.0 
mL HN0 3 , and titr. with dil. std AgN0 3 soln as above. 

Ppm CI in sample = (1/ W /IA>) x C x 10, 
where t/ w = mL std AgN0 3 used by sample, V s = mL std AgN0 3 
used by std CI soln, and C = 0.5 mg CI in 5 mL std CI soln used. 

9.046 Total Acids — Official Final Action 

Neutze ca 250 mL boiled H 2 in porcelain evapg dish (185 
mm dish is convenient). Add 25 mL sample and titr. with 0.1/V 
NaOH, using ca 2 mL phthln. 

9.047 Fixed Acids— Official Final Action 

Evap. 25-50 mL sample to dryness in Pt dish on steam bath 
and dry 30 min in oven at 100°. Dissolve and transfer residue 
with several portions of neut. alcohol of ca same proof as 
sample, using 25-50 mL in all, to porcelain dish contg ca 250 
mL neutzd boiled H 2 0. Titr. with 0.1/V NaOH, using 10 mL buret 
graduated in 0.05 mL, and ca 2 mL phthln. 

9.048 Volatile Acids — Official Final Action 

Volatile acids = total acids from 9.046 - fixed acids from 
9.047. 



9.049 



Esters and Aldehydes — Official Final Action 



Reagents 



(a) Sodium thiosuifate std soln— -0.05/V. Prep, by dilg 0.1/V 
soln, 50.037. 

(b) Iodine soln. — Approx. 0.05/V. 

(c) Sodium bisulfite soln. — Approx. 0.05/V. (Deterioration is 
retarded if soln contains ca 10% alcohol; do not use after ca 1 
week.) 



Transfer 100 mL distillate to 500 mL flask, neutze free acid, 
add measured excess 0.1/V NaOH, connect flask with air-cooled 
condenser ca 60 cm long, heat 2 hr on steam bath, let cool, and 
titr. excess alkali. Reject detns in which excess 0.1/V alkali is <2 
mL, or is >10 mL. Calc. esters as EtOAc. Correct for blank detn 
performed on 100 mL 50% alcohol (absolute alcohol-H 2 0, 1+1). 



9.052 Determination of Aldehyde 

{Indirect Method) 

Place remainder of distillate from 9.050 in 500 mL flask, add 
ca 100 mL H 2 and excess NaHS0 3 soln, and let stand ca 30 
min, shaking occasionally. (Excess NaHS0 3 should be equiv. of 
ca 25 mL I soln.) Add excess I soln, and titr. this excess with std 
Na 2 S 2 3 soln. Run blank contg same vols of I soln and bisulfite 
soln as used in sample. Difference between titrns in mL Na 2 S 2 3 
soln x 1.1 = mg acetaldehyde in sample. 



Esters 

Spectrophotometric Method [12) — Official Final Action 

9.053 Principle 

Esters react quant, with H 2 NOH in alk. soln to form a hydrox- 
amic acid which, after acidification, forms colored complex with 
ferric ions. Ester concn is proportional to A at 525 nm at const 
alcohol concn and can be calcd from either of 2 std curves: (/) 
At constant proof . — Plot>4 against known EtOAc concn in 100° 
proof spirits (or any other convenient const proof); or {2) At any 
actual proof. — Plot>4 of EtOAc/g against proof and calc. concn 
from this proof factor. 



9.054 



Reagents 



(a) Hydrochloric acid.-— AN. Dil. 333 mL HCl to 1 L with H 2 0. 

(b) Ferric chloride soln.— 0.37 M. Dissolve 50 g FeCI 3 .6H 2 in 
ca 400 mL H 2 in 500 mL vol. flask. Add 12.5 mL 4/V HCl and dil. 
to vol. with H 2 0. 

(c) Hydroxylamine hydrochloride soln. — 2M. Dissolve 69.6 g 
H 2 NOH.HCI in H 2 in 500 mL vol. flask and dil. to vol. with H 2 0. 
Store in refrigerator. 

(d) Sodium hydroxide soln.— Z.bN. Dissolve 70 g NaOH in ca 
400 mL H 2 in 500 mL vol. flask. Cool, and dil. to vol. with H 2 0. 



9.055 



Preparation of Ethyl Acetate Standard Solutions 



(a) Stock soln No. 7 for std curve.— 0.333 g EtOAc/L in 100° 
proof spirits. Weigh 0.1667 g EtOAc in weighing bottle and 
transfer quant, to 500 mL vol. flask with 100° proof spirits. Dil. 
to vol. with 100° proof spirits at room temp. 

(b) Std solns for std curve. — To series of five 100 mL vol. 
flasks add, from pipet or buret, 0.0, 15.0, 30.0, 45.0, and 60.0 mL 
stock soln No. 1. Dil. to vol. with 100° proof spirits and mix. Stds 
contain 0.0, 5.0, 10.0, 15.0, and 20.0 g EtOAc/100 L, resp. 

(c) Stock soln No. 2 for proof factor determination. — 0.500 g 
EtOAc/L H ? 0. Weigh 0.2500 g EtOAc in weighing bottle and 



AOAC Methods (1980) 



Aldehydes 



153 



transfer quant, to 500 ml_ vol. flask with H 2 0. Dil. to vol. with 
H 2 and mix. 

(d) Stock soln No. 3 for proof factor determination . — 0.500 g 
EtOAc/L 192° proof spirits. Prep, as in (c), using 192° proof 
spirits for transfer and diln. 

(e) Proof factor std solns.— {1) 0-50° stds.—lo 6 sep. 250 mL 
vol. flasks add 50.0 mL EtOAc stock soln No. 2. Using graduate, 
add 0, 13, 20, 39, 53, and 66 mL 192° proof spirits. Dil. each 
nearly to vol. with H 2 0, mix, and cool to room temp, before dilg 
to vol. with H 2 0. Std solns contain 10 g EtOAc/100 L in ca 0, 10, 
20, 30, 40, and 50° proof spirits. (2) 60-192° stds — To 9 sep. 250 
mL vol. flasks, add 50.0 mL EtOAc stock soln No. 3. Using 
appropriate graduate, add 29, 55, 81, 108, 134, 161, 174, 187, 
and 200 mL 192° proof spirits, resp. Dil. nearly to vol. with H 2 0, 
mix, and cool to room temp, before dilg to vol. with H 2 0. Std 
solns contain 10 g EtOAc/100 L in ca 60, 80, 100, 120, 140, 160, 
170, 180, and 192° proof spirits, resp. 

Det. exact proof of solns by std method, e.g. hydrometer. 

9056 Preparation of Samples 

Analyze whisky distillates, spirits, and colorless gin samples 
directly. Distil colored or turbid samples as in 9.050. If sample 
contains >20 g ester/100 L, dil. with H 2 to ester concn of 5-20 
g/100 L. 

9.057 Determination 

(Mix all solns by swirling to avoid formation of bubbles.) 

Just before use, prep, stock soln of reaction mixt. by combining 
5.0 mL H 2 NOH.HCI and 5.0 mL 3.5/V NaOH for each std and 
sample soln. Discard after 6 hr. 

Prep. ref. soln by pipetting 4 mL reaction mixt. and 2 mL AN 
HCI into 25 x 200 mm test tube. Mix and add 2.0 mL sample. 
Same ref. soln may be used for series of samples of different 
ester content, but they must have same proof. 

Pipet 2 mL sample and 4 mL reaction mixt. into another 25 x 
200 mm test tube. Mix and let react 1-20 min. Pipet in 2 mL 4/V 
HCI and mix. 

To ref. soln, pipet in 2 mL FeCI 3 soln. Rinse ref. cell twice with 
this soln, fill cell, and place in cell holder. This ref. soln may be 
used for 1 day if tightly capped; otherwise refill periodically to 
avoid evapn error. 

To sample soln, pipet in 2 mL FeCI 3 soln and mix. Complete 
reading of each sample before proceeding to next. Rinse sample 
cell twice, fill cell, and place in cell holder. Read A at 525 nm 
immediately, since color of sample fades rapidly. If instrument 
has single cell or tube, use same cell or tube for both ref. and 
sample. Calc. or obtain AA = /4 sampte - A ret . 

9.058 Preparation of Standard Curve 

Analyze std solns, 9.055(b), as in 9.057. Plot AA against EtOAc 
concn (g/100 L at 100° proof). {Note: Std curve need not be 
repeated for each analysis. Check periodically and repeat if new 
instrument or reagents are used.) 

9.059 Preparation of Proof Factor Curve 

Analyze std solns, 9.055(e), as in 9.057. Plot AA/g EtOAc 
against proof in the 15 solns (0-192° proof). See Note, 9.058. 

To calc. ester content of samples, read A/g value from proof 
factor curve at sample proof. If sample was dild, use dild proof 
in calcn. Divide observed A by A/g to obtain g EtOAc/100 L. 
Correct for sample diln, if necessary. To express as g/100 L at 
100° proof, multiply above ester value by ratio: 100/sample 
proof. 



Aldehydes — Official Final Action 
Method I {13) 

(Applicable to ext-free spirits — brandy and wine spirits) 

9.060 Reagents 

(a) Potassium metabisuifite so/n. — Dissolve 15 g K 2 S 2 5 in 
H 2 0, add 70 mL HCI, and dil. to 1 L with H 2 0. Bisulfite titer of 10 
mL soln should be ^24 mL 0.1/V I soln. 

(b) Phosphate-EDTA soln.— Dissolve 200 g Na 3 P0 4 .12H 2 (or 
188 g Na 2 HP0 4 .12H 2 + 21 g NaOH; or 72.6 g NaH 2 P0 4 .H 2 + 
42 g NaOH; or 71.7 g KH 2 P0 4 + 42 g NaOH) and 4.5 g Na 2 H 2 EDTA 
in H 2 and dil. to 1 L 

(c) Dilute hydrochloric ac/d.—Dil. 250 mL HCI to 1 L with H 2 0. 

(d) Sodium borate soln.— Mix 100 g H 3 B0 3 with 170 g NaOH 
and dil. to 1 L with H 2 0. 



9.061 



Total Aldehydes 



Pipet 50 mL sample (contg ^30 mg acetaldehyde), reduced 
to ca 100° proof, or 25 mL high proof sample and 25 mL H 2 0, 
into 750 mL or 1 L erlenmeyer contg 300 mL boiled or deaerated 
H 2 and 10 mL K 2 S 2 5 soln. Stopper flask, swirl to mix, and let 
stand 15 min. Add 10 mL phosphate-EDTA soln. (pH should be 
7.0-7.2. if not, adjust pH by adding HCI or NaOH soln to K 2 S 2 5 
soln and start with new sample.) Stopper flask, swirl, and let 
stand addnl 15 min. Add 10 mL HCI, (c) (when analyzing series, 
make complete detn on first sample before adding acid to next), 
and ca 10 mL fresh 0.2% starch indicator. Swirl to mix. Add 
enough ca 0.1/V I soln to just destroy excess bisulfite and bring 
soln to light blue end point. 

Add 10 mL Na borate soln, and rapidly titr. liberated bisulfite 
with 0.05/V I soln from 10 mL buret (or 0.02/V I soln from 25 mL 
buret) to same light blue end point as above, swirling gently 
and continuously, avoiding direct sunlight (pH should be 
8.8-9.5. If necessary, adjust by adding HCI or NaOH soln to Na 
borate soln and start with fresh sample.) 

mg CH 3 CHO/100 mL = mL I soln x normality I soln x 22.0 x 
100/mL sample. 

9.062 Free Aldehydes 

Pipet identical sample as in 9.061 into 750 mL or 1 L erlen- 
meyer contg 300 mL boiled or deaerated H 2 and 10 mL each 
K 2 S 2 5 and phosphate-EDTA solns. Stopper flask, swirl, and let 
stand 15 min. Proceed as in 9.061, beginning "Add 10 mL HCI, 
(c) . . ." 



Method II 

(Applicable to spirits contg ext — aged in wood) 

9.063 Free Aldehydes 

Pipet 50 mL sample (contg =£30 mg acetaldehyde), reduced 
to 80-100° proof, if necessary, into 500 mL distg flask, add 50 
mL satd borax soln, and distil ca 50 mL into 750 mL or 1 L 
erlenmeyer contg 300 mL H 2 and 10 mL each K 2 S 2 5 and 
phosphate-EDTA solns. (pH should be 7.0-7.2. If necessary, 
adjust by adding HCI or NaOH soln to K 2 S 2 5 soln and start with 
fresh sample.) Proceed as in 9.061, beginning "Add 10 mL HCI, 
(c) . . ." 



9.064 



Aldehydes as Acetal 



Transfer 200 mL sample measured at std temp, in vol. flask 
to 500 mL distg flask, and rinse vol. flask 2-3 times with small 
amts H 2 into distg flask. Add 50 mL satd borax soln and distil, 



154 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



slowly at first nearly 200 ml_ into same vol. flask contg 2-3 mL 
H 2 and immersed in ice bath. Bring distillate to vol. at same 
temp, used for measuring sample. 

Det. total aldehydes (including acetal) as in 9.061. Det. free 
aldehydes as in 9.062. Total aldehydes - free aldehydes = 
combined aldehydes equiv. to acetal as mg CH 3 CHO/100 mL. 
Alternatively, combined aldehydes as acetal/ 100 mL = (com- 
bined aldehydes equiv. to acetal as mg CH 3 CHO/100 mL) x 2.68. 

9.065 Total Aldehydes 

Report total aldehydes as sum of free aldehydes, 9.063, and 
combined aldehydes equiv. to acetal, 9.064. 



Fusel Oil — Official Final Action 
Method I [14) 



9.066 



Reagents 



(a) p-Dimethylaminobenzaldehyde {DMAB) soln. — In 100 mL 
vol. flask dissolve 1 g DMAB in mixt. of 5 mL H 2 S0 4 and 90 mL 
H 2 0, and dil. to vol. with H 2 0. 

(b) Isobutyl alcohol. — Eastman Kodak Co. 303 (highest purity 
for fusel oil assay). 

(c) Isoamyl alcohol. — Eastman Kodak Co. X-18, isopentyl al- 
cohol (highest purity for fusel oil assay). 

(d) Ethyl alcohol— Redistd middle 50% fraction. 

(e) Synthetic std fusel oil. — Weigh 2 g std isobutyl alcohol 
and 8 g std isoamyl alcohol into 1 L vol. flask and dil. to vol. 
with H 2 0. Pipet two 10 mL portions into 100 mL vol. flasks and 
dil. to vol., one with H 2 and other with alcohol. Prep, working 
stds for products in range of 0-170 proof contg 1.0-6.0 g 
synthetic fusel oil/100 L by dilg 1.0-6.0 mL aliquots of aq. std 
soln to 100 mL with ale. soln of proof expected for dild sample 
when pipetted into analysis tube. Prep, similar working stds for 
products in range of 170-190 proof by dilg 1.0-6.0 mL aliquots 
of ale. std soln to 100 mL with ale. soln of proof of sample or 
its diln. 

When 6 mL synthetic std dild with 190 proof alcohol is 
analyzed, >4 should be 0.83±0.03 at 530 nm. 

9.067 Preparation of Samples 

(Aged, blended and rectified products, whiskies, brandies, rums, 
vodka, and liqueurs require distn prior to analysis.) 

Determination of true proof of sample. — Det. alcohol as in 
9.013. 

For samples contg >6 g fusel oil/ 100 L, dil. distd sample with 
H 2 to concn of 2.0-5.0 g fusel oil/ 100 L. Dil. 5 mL brandy, rum, 
or blended whisky to 100 mL; dil. 5 mL heavy brandy, rum, or 
straight whisky to 250 mL. 



9.068 



Determination 



Pipet 2 mL aliquots of sample or dild sample, distd, if 
necessary, 2 mL H 2 (for reagent blank), and 2 mL aliquots of 
stds into 15 x 150 mm g-s or covered test tubes. Stopper or 
cover tubes, and place in rack, then in ice bath. Pipet 1 mL DMAB 
soln into each tube, shake, and replace in ice bath for 3 min. 
With tubes still in ice bath, add 10 mL chilled H 2 S0 4 from buret 
down side of tubes. Shake tubes individually and replace in ice 
bath for 3 min. Transfer rack of tubes from ice bath to boiling 
H 2 bath and boil 20 min. Transfer tubes to ice bath for 3-5 
min, then to room temp. bath. Read %T of developed color of 
samples and stds on spectrophtr at 538-543 nm against reagent 
blank as ref. (Use same wavelength for both stds and unknowns.) 



Plot g fusel oil in std samples/100 L on linear scale as abscissa 
against %T as ordinate on log scale of semilog paper. Convert 
%T of samples to g fusel oil/100 L from std curve. If diln was 
used, multiply g fusel oil/100 L found by diln factor to obtain g 
fusel oil/100 L in original sample. Analyze 2 levels of stds with 
each series of unknowns. 

Precision expected: Whisky and brandy, ±5%; rum, ±8%; 
gin, vodka, and spirits, ±0.4 g/100 L. 



Method II [15) 



9.069 



Reagents 

(a) Color reagent. — Dissolve 1 g Na salt of 4-hydroxybenzal- 
dehyde-3-sulfonic acid in H 2 0, dil. to vol. in 25 mL vol. flask with 
H 2 0, and filter. 

(b) Fusel oil std so/ns— Weigh 2 g isobutyl alcohol, 9.066(b), 
and 8 g isoamyl alcohol, 9.066(c), into 1 L vol. flask and dil. to 
vol. with 50% alcohol. Dil. 0, 1, 2, 3, 5, 10, and 15 mL portions 
to vol. with 50% alcohol in 100 mL vol. flasks (0.0, 0.1, 0.2, 0.3, 
0.5, 1.0, and 1.5 g fusel oil/L). Std soln contg 1.0 g fusel oil/L 
should give A of ca 0.4 at 445 nm in 9.071. 

(c) Alcohol.— 50%. Free of fusel oil (ACS). 



9.070 



Preliminary Distillation 



Add 20 mL H 2 to 50 mL sample and distil, slowly at first, 
collecting ca 50 mL in 50 mL vol. flask. Dil. to vol. with H 2 0. (For 
samples known to contain >150 g fusel oil/100 L, use 25 mL 
sample plus 45 mL H 2 0.) 



9.071 



Determination 



To dry 10 mL vol. flask, add 0.1 mL distillate from serological 
blow-out pipet. (Pipet should have pointed tip and should be 
thoroly cleaned (chromic acid plus H 2 rinse) before use. Rinse 
pipet several times with distillate, and wipe end dry. Bring liq. 
to line while holding tip to outside surface of vol. flask. Then 
insert end to bottom of vol. flask and release sample. After 
draining pipet, hold flask at 45° angle and blow out pipet.) 

Add 0.1 mL color reagent from 1 mL buret graduated in 0.01 
mL; then add 2 mL H 2 S0 4 from 50 mL buret. Mix, and place 
unstoppered flask in H 2 bath at room temp. (250 mL beaker is 
convenient). Bring to bp and boil 30 min. Let cool, dil. to vol. 
with H 2 S0 4 , and det. A at 445 nm against H 2 on Beckman DU 
spectrophtr, or equiv. instrument. 



9.072 



Blank and Standards 



Develop color of 0.1 mL 50% alcohol and 0.1 mL portions of 
std solns. Use same 0.1 mL pipet for blank, stds, and sample. 
Subtract A of 50% alcohol blank from A of sample and stds. 



9.073 



Calibration Curve or Factor 



Plot corrected>4 of stds against concn. (Straight line is obtained 
up to ca 150 g fusel oil/ 100 L; above this value, curve flattens. 
Concn of color reagent may be increased, but it is best to dil. 
distd sample so that A is <0.6.) Calc. or obtain A' [A of 1.0 g 
fusel oil/L) from curve. 



9.074 



Calculations 



g fusel oil/100 L = 100 A x D/A' 
where 100 = diln of 0.1 mL sample to lOmLincolordevelopment, 
A ~ corrected A of sample, A' = factor, 9.073, and D = diln of 
sample before distn. 



AOAC Methods (1980) 



Higher Alcohols 



155 



Higher Alcohols (n-Propyl Alcohol, Isobutyl Alcohol, and 
Isoamyl Alcohol) and Ethyl Acetate — Official Final Action 

Gas Chromatographic Method [16) 

9.075 Apparatus 

(a) Gas chromatograph.— Equipped with flame ionization de- 
tector. (7) Column. —23% Carbowax 1500 (w/w) on Chromosorb 
W {60-80 mesh, acid-washed). Weigh 9 g Carbowax 1500 into 
250 mL beaker and mix with H 2 on steam bath. Weigh 30 g 
Chromosorb W in 250 mL beaker and combine with Carbowax 
soln in large flat-bottom Pyrex glass baking dish or flat-bottom 
polyethylene container (ca 20 x 25 cm). Add H 2 to just cover 
solid support and mix thoroly. Evap. H 2 with frequent stirring 
in hood. (Gentle steam may be applied to hasten evapn.) After 
evapn of H 2 0, heat coated support ca 2 hr in 100° oven. 

Pack 2.4 m (8') x V£" od Cu tubing tightly and evenly by 
repeated tapping, and condition in column oven at 150° with He 
flow rate of 150 mL/min until steady baseline is observed at 
attenuation 1x at operating parameters {ca 24 hr). 

(2) Approximate parameters. — Column temp. 70° (isother- 
mal); detector and inlet temp. 1 50°; He carrier flow 1 50 mL/min. 

Optimum operating conditions vary with column and instru- 
ment, and must be detd by using std solns. Adjust parameters 
for max. peak sharpness and optimum sepn. With high level 
std, /7-PrOH should give almost complete baseline sepn from 
EtOH. 

(b) Syringe. — 10 /u,L, Hamilton Co. No. 701, or equiv. 



9.076 



Reagents 



(a) Isobutyl a Icohoi.—see 9.066(b). 

(b) Isoamyl alcohol. — See 9.066(c). 

(c) n-Propyl alcohol. — Redistd, reagent grade. 

(d) Ethyl acetate. — Redistd, reagent grade. 

(e) n-Butyl alcohol. — Redistd, reagent grade. 

(f) n-Butyf alcohol internal std solns.— {1) High level. — Dil. 10 
mL/7-BuOHto 100 mL with 40% alcohol. (1 mL added to 100 mL 
sample or std is equiv. to ca 81 g /?-BuOH/100 L.) (2) Low 
level.— Dil. 1 mL n-BuOH to 200 mL with 95% alcohol. (1 mL 
added to 100 mL sample or std is equiv. to ca 4.1 g/100 L.) 

(g) n-Propyl alcohol, isobutyl alcohol, isoamyl alcohol, and 
ethyl acetate high level std solns.— (1) Stock soln.— Accurately 
weigh 1 mLn-PrOH, 1 mL isobutyl alcohol, 2 mL isoamyl alco- 
hol, and 1 mL EtOAc into 100 mL vol. flask and dil. to vol. with 
40% alcohol. (2) Intermediate soln. — Dil. 10 mL stock soln to 
200 mL with 40% alcohol. [3) Working soln.— (Approx. 40.2, 
41.1, 81.2, and 45.1 g/100 L n-PrOH, isobutyl alcohol, isoamyl 
alcohol, and EtOAc, resp.) Dil. 5 mL stock soln to 100 mL with 
40% alcohol. Add 1 mL high level n-BuOH internal std soln, 
(f)(7), and mix. Prep, fresh weekly. 

(h) n-Propyl alcohol, isobutyl alcohol, isoamyl alcohol, and 
ethyl acetate low level working solns.— {Approx. 2.0, 2.1, 4.1, 
and 2.3 g/100 L n-PrOH, isobutyl alcohol, isoamyl alcohol, and 
EtOAc, resp.) Dil. 5 mL high level intermediate soln, (g)(2), to 
100 mL with 95% alcohol. Add 1 mL low level internal std soln, 
(f)(2), and mix. Prep, fresh weekly. 

Prep, std soln of ca same concn as sample if latter differs 
grossly from appropriate (high or low level) std. 



9.077 



Determination 



Make preliminary injection of 10 ju,L sample to det. absence 
of n-BuOH. (If present, subtract its amt from total /7-BuOH 
(original and internal std) content.) Add 1 mL internal std soln, 
(f) (high or low level, depending on higher alcohol and EtOAc 



concn), to 100 mL sample in vol. flask, and chromatograph 10 
(iL aliquots of sample and std solns in triplicate. 

Measure peak hts of /?-PrOH, isobutyl alcohol, isoamyl alcohol, 
and EtOAc to nearest 0.05 cm and calc. peak ht ratio of each to 
/7-BuOH (internal std) in sample and std solns. (For more accurate 
detn of isoamyl alcohol, use peak areas.) 

X = H x S/H', where X = concn of higher alcohol or EtOAc 
in sample (g/100 L); H = peak ht (or area for isoamyl alcohol) 
ratio of higher alcohol or EtOAc to /?-BuOH in sample; H' = 
peak ht (or area for isoamyl alcohol) ratio of higher alcohol or 
EtOAc to n-BuOH in std; 5 = concn of higher alcohol or EtOAc 
in std (g/100 L). 

Sum of isoamyl and isobutyl alcohol concns is ca equiv. to 
fusel oil concn as detd in 9.069-9.074. 



Aiternative Method (17) 



9.078 



Apparatus 



Gas chromatograph.— Equipped with flame ionization detec- 
tor. (/) Column, —2% glycerol and 2% 1,2,6-hexanetriol (Aldrich 
Chemical Co.) (w/w) on Gas-Chrom R (100-120 mesh, non-acid- 
washed) (Applied Science Laboratories, Inc.). Weigh 0.2 g each 
of glycerol and 1,2,6-hexanetriol into 50 or 100 mL beaker and 
dissolve by stirring with MeOH. Weigh 9.6 g Gas-Chrom R in 
evapg dish (top diam. ca 100-125 mm), moisten with MeOH, 
and add soln of stationary phases. Mix thoroly, and gently heat 
mixt. on steam bath, stirring continuously. When material 
appears to be dry (light pink), continue heating and occasional 
stirring to ensure complete removal of solv. Store in capped jar. 
Pack 3 m (10') x 1 / 8 " od Cu or stainless steel tube (0.030" wall; 
if steel is used, rinse inside several times with acetone to remove 
manufacturing oils, and air dry) with prepd support, using 
vibrator to ensure complete packing of column. Condition 
overnight in 80° column oven with He flow rate of 10-25 mL/min 
and detector end of column disconnected. 

(2) Approximate parameters. — Column, injector, and detector 
temps (°) — 80, 100, and 125, resp.; gas flows (mL/min)— He 
carrier and H 25, air 250-400; attenuation 64x. 

Optimum operating conditions vary with column and instru- 
ment and must be detd by using std solns. Adjust parameters 
for max. peak sharpness and optimum sepn. Analysis is com- 
plete in ca 11 min. 

9.079 Reagents 

(Use absolute alcohol thruout when alcohol is specified.) 

(a) n-Propyl alcohol.— Reagent grade. 

(b) Isobutyl alcohol.— {Fisher Scientific Co., Certified Reagent 
No. A-379.) 

(c) Amy! alcohol. — (Gallard-Schlesinger Chemical Manufac- 
turing Co., 584 Mineola Ave., Carle Place, NY 1 1514, AnalaR No. 
10038.) Mixt. of active-amyl and isoamyl alcohols, ca 22 and 
78%, resp. Concn of 2 isomers varies from batch to batch. Det. 
composition of reagent by 9.080. Measure areas of 2 peaks by 
triangulation (ht x width at half-ht), and obtain concn of each 
by dividing area of each peak by sum of both peak areas. 

(d) 3-Pentanol internal std soln.— 40.76 mg/mL (Aldrich 
Chemical Co., Inc., No. P802-5). Prep, soln contg 10 mL reagent 
in 200 mL alcohol-H 2 (1+1). 

(e) Ethyl acetate. ~~{F\sher Scientific Co., Certified Reagent No. 
E-145.) 

(f) n-Propyl alcohol, isobutyl alcohol, and a my I alcohol std 
solns. — Prep. 3 or 4 std solns contg varying amts alcohols as 
follows: Into tared 100 mL vol. flasks contg alcohol-H 2 (1+1), 
pipet fusel alcohols and weigh after addn of each component. 



156 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



Proportions of fusel alcohols in each std soln should vary so 
that desired concn range of each is represented in random 
manner in series of std solns. Suggested amts: 0.25-1.5 mL n- 
PrOH, 1.0-2.5 mL isobutyl alcohol, and 2.0-5.0 mL amyl alcohol. 
Dil. each to vol. with alcohol-H 2 (1 + 1). 

(g) n-Propyl alcohol, isobutyl alcohol, and amyl alcohol work- 
ing std solns. — Dil. 10 mL each std soln and 2.0 mL 3-pentanol 
internal std soln to 200 mL with alcohol-H 2 (1 + 1) (1:20 diln). 

(h) Ethyl acetate std solns. — Prep. 3 or 4 std solns contg 0-0.5 
g/L (0-50 g/100 L) in H 2 or alcohol-H 2 (1 + 1). Use for prepg 
direct std curve by plotting peak ht (mm) against concn in g/100 
L. 



9.080 



Determination 



Pipet 10 mL sample into convenient vessel (e.g., 1 oz French 
sq glass bottle with screw cap), add, by pipet (0.2 mL pipet 
graduated in 0.01 mL), 0.1 mL 3-pentanol internal std soln, and 
mix. Inject 2 /aL sample and working std solns. Measure peak 
ht of each component in working std soln and calc. peak ht ratio 
of each to internal std. Calc. concn ratio of each by dividing wt 
of component by that of internal std. (Proportion of active-amyl 
and isoamyl alcohols in mixt. must be taken into consideration 
in calcns of actual wts of each isomer in working std solns.) 

Plot concn ratios (horizontal axis) against peak ht ratios 
(vertical axis) for each higher alcohol in all working stds to 
obtain family of curves. For EtOAc, plot peak ht directly against 
concn. 

Similarly, measure peak ht of each component in sample and 
calc. peak ht ratios. Read concn ratios of all alcohols, using 
proper std curve. Multiply concn ratio of each fusel alcohol in 
sample by 40.76 to obtain g/100 L. New std curves need be 
prepd only when new instruments, parameters, or stds are used. 



Furfural (75)— Official Final Action 

9.081 Reagent 

Furfural std soln. — Redistil furfural thru short packed fraction- 
ating column at atm. pressure, and collect fraction boiling at 
161.2° (uncorrected). Weigh 1 mL redistd furfural into 100 mL 
vol. flask and dil. to vol. with alcohol. Pipet 5 mL of this soln into 
500 mL vol. flask and dil. to vol. with 50% alcohol (concn, ca 1 1 6 
mg/L). Coned soln retains strength, but dil. soln does not. 

9.082 Determination 

Pipet 25 mL distd spirits into volatile acid distn flask, Fig. 
9:03, with f joints and steam distil until 200 mL collects. If haze 
is present in distillate, dil. with known vol. alcohol. Det.4 at 277 
nm. 

Det. A of std solns of furfural contg 0, 1, 2, 3, 4, and 5 mg 
furfural/L. Plot std curve or calc. av. A of 1 mg furfural/L, A' (ca 
0.15). 

mg Furfural/L = {A/A') x F t 
where F is diln factor (vol. final soln on which A is detd/vol. 
sample). 

Detection of Acetone, Other Ketones, 

Isopropanol, and Tertiary Butyl Alcohol 

Official Final Action 

9.083 Reagent 

Mercuric sulfate soln. — Mix 5 g yellow HgO with 40 mL H 2 
and add, with stirring, 20 mL H 2 S0 4 and 40 mL H 2 0. Stir until 
completely dissolved. {Caution: See 51.079.) 



Jem(nppioi) /Mt«llh ttepptr) 




l&crc Upproi) 



FIG. 9:03— Steam distillation flask 



9.084 



Determination 



To 2 mL distillate, 9.050, add 3 mL H 2 and 10 mL HgS0 4 
soln. Heat on boiling H 2 bath 3 min. White or yellow ppt 
forming within 3 min indicates presence of acetone, other 
ketones, or tert-BuOH. Disregard any ppt forming after 3 min on 
boiling H 2 bath. 

If no ppt forms, test for isopropanol as follows: Place 8 g Cr0 3 
in 100 mL Kohlrausch flask, and add 15 mL H 2 and 2 mL H 2 S0 4 . 
Connect flask with reflux condenser and add 5 mL sample very 
slowly thru condenser. Reflux 30 min; then cool and distil 2 mL, 
collecting distillate in 10 mL graduate. Add 3 mL H 2 and 10 mL 
HgS0 4 soln, and proceed as above. 

9.085 Sugars — Official Final Action 
See Chapter 31. 

Methanol — Official Final Action 
Chromotropic Acid Colorimetric Method {19) 



9.086 

(a) Potassium permanganate soln.- 



Reagents 

-Dissolve 3.0 g KMn0 4 



and 15.0 mL H 3 P0 4 in 100 mL H 2 0. Prep, monthly. 

(b) Sodium salt of chromotropic acid {sodium 1,8-dihy- 
droxynaphthalene-3,6-disulfonate) soln. — 5% aq. soln. Filter if 
not clear. Prep, weekly. Either acid or salt may be used. 

9.087 Purification of Chromotropic Acid 

If A of blank is >ca 0.05, purify reagent as follows: 
Dissolve 10 g chromotropic acid or its salt in 25 mL H 2 0. (Add 
2 mL H 2 S0 4 to aq. soln of salt to convert it to free acid.) Add 50 
mL MeOH, heat just to bp, and filter. Add 100 mL isopropanol 
to ppt free chromotropic acid. (Add more isopropanol to increase 
yield of purified acid.) 

9.088 Preparation of Sample 

Dil. or adjust sample to total ale. concn of 5-6%. Using 50 mL 
sample, distil thru simple still, collecting 40 mL distillate. Dil. to 
50 mL with H 2 0. {If alcohol has been detd previously, distillate 
may be adjusted to 5-6% ale. concn and used for this test.) If 



AOAC Methods (1980) 



Methanol 



157 



>0.05% MeOH by vol. is present dil. to ca that concn with 5.5% 
alcohol. For samples contg <0.05% MeOH, measure 200 mL 
into efficient fractionating stilt, place system under total reflux 
15 min, and then slowly distil at high rate of reflux (s=20:1). 
Collect 10 mL distillate and dil. to 160 mL with H 2 0. 



9.089 



Determination 



Pipet 2 mL KMn0 4 soln into 50 mL vol. flask. Chill in ice bath, 
add 1 mL chilled dild sample, and let stand 30 min in ice bath. 
Decolorize with little dry NaHS0 3 and add 1 mL chromotropic 
acid soln. Add 15 mL H 2 S0 4 slowly with swirling and place in 
hot (60-75°) H 2 bath 15 min. Cool, add enough H 2 to bring 
approx. to 50 mL mark, mix, and dil. to vol. with H 2 at room 
temp. Read A at 575 nm against reagent blank of 5.5% alcohol 
treated similarly. Treat std MeOH soln contg 0.025% by vol. 
MeOH in 5.5% alcohol simultaneously in same manner, and 
read A', (Temp, of std and sample should be within 1° since 
temp, affects A.) 

% MeOH in sample = {A/ A') x 0.025 x F, 
where F ~ diln factor of sample. 

Example: Sample was dild 25 x; A of sample - 0.421; A of 
std MeOH - 0.368. Then (0.421/0.368) x 0.025 x 25 = 0.715% 

(If color of sample is too intense, dil. with H 2 S0 4 -alcohol blank 
prepd as above. Not more than 3-fold diln is permitted, as ratio 
of chromotropic acid to HCHO is too low if diln is greater.) 

9.090 immersion Refractometer Method [20) 

Det. Zeiss immersion refractometer reading at 17.5° of distillate 
obtained in detn of alcohol. If, on ref. to table, 9:03, refractometer 
reading shows sp gr agreeing with that obtained in alcohol detn, 
9.013, it may be assumed that MeOH is absent. Low refractom- 



eter reading indicates presence of appreciable amt of MeOH. If 
absence from the soln of refractive substances other than H 2 
and the alcohols is assured, this difference in refraction is 
conclusive evidence of presence of MeOH. 

Addn of MeOH to alcohol decreases refractive index in direct 
proportion to amt added; hence, quant, calcn is made by 
interpolation in Table 9:03 of figures for pure alcohol and MeOH 
of same sp gr as sample. 

Example. — Distillate has sp gr at 15.56° of 0.9625 and refrac- 
tometer reading at 17.5° of 43.1. By interpolation in Table 9:03, 
readings for alcohol and MeOH at this gravity are 65.2 and 31.7, 
resp., and difference is 33.5; 65.2 - 43.1 - 22.1; (22.1 -r- 33.5) 
x 100 = 66.0, showing 66.0% of total alcohol present is MeOH. 

Gas Chromatographic Method [27) 

9.091 Apparatus 
See 9.075. 

9.092 Reagents 

(a) Alcohol.— USP, MeOH-free. 

(b) Methanol stock soln.— Dil. 10 mL MeOH, 99.9 mol % 
(Fisher Scientific Co., A-936, or equiv.) to 100 mL with 40% 
alcohol. 

(c) n-Butyl alcohol internal std stock soln.—D\\. 10 mLn- 
BuOH, 99.9 mol % (Fisher Scientific Co., A-384, or equiv.) to 100 
mL with 40% alcohol. 

(d) Methanol std soln.— 0.050% MeOH plus 0.030% n-BuOH 
internal std. Fill 100 mL vol. flask to ca 99 mL with 40% alcohol 
and add, by syringe, 500 ju,L MeOH stock soln, (b), and 300 /xL 
n-BuOH stock soln, (c). Mix and dil. to vol. with 40% alcohol. 
Mix again. 



Table 9:03 



Scale readings on Zeiss immersion refractometer at 17.5°, corresponding to specific gravities of ethyl and methyl 

alcohol solutions 



Sp. Gr. 


Scale Readings 


Differences 


Sp. Gr. 
15.56° 


Scale Read 


ngs 




15.56° 


Ethyl 
Alcohol 


Methyl 
Alcohol 


Ethyl 

Alcohol 


Methyl 
Alcohol 




15.56° 


15.56 


Differences 


1.0000 


15.0 


15.0 


0.0 


0.9720 


51.5 


27.0 


24.5 


.9990 


15.8 


15.3 


0.5 


.9710 


53.0 


27.5 


25.5 


.9980 


16.6 


15.6 


1.0 


.9700 


54.6 


28.1 


26.5 


.9970 


17.5 


15.9 


1.6 


.9690 


56.1 


28.7 


27.4 


.9960 


18.5 


16.2 


2.3 


.9680 


57.6 


29.2 


28.4 


.9950 


19.4 


16.5 


2.9 


.9670 


59.1 


29.6 


29.5 


.9940 


20.4 


16.9 


3.5 


.9660 


60.6 


30.1 


30.5 


.9930 


21.4 


17.2 


4.2 


.9650 


62.0 


30.6 


31.4 


.9920 


22.5 


17.5 


5.0 


.9640 


63.3 


31.0 


32.3 


.9910 


23.6 


17.9 


5.7 


.9630 


64.6 


31.5 


33.1 


.9900 


24.7 


18.2 


6.5 


.9620 


65.8 


31.9 


33.9 


.9890 


25.9 


18.6 


7.3 


.9610 


67.0 


32.4 


34.6 


.9880 


27.1 


19,0 


8.1 


.9600 


68.1 


32.8 


35.3 


.9870 


28.4 


19.5 


8.9 


.9590 


69.2 


33.3 


35.9 


.9860 


29.6 


19.9 


9.7 


.9580 


70.2 


33.7 


36.5 


.9850 


31.0 


20.4 


10.6 


.9570 


71.2 


34.1 


37,1 


.9840 


32.4 


20.8 


11.6 


.9560 


72.1 


34.5 


37.6 


.9830 


33.8 


21.3 


12.5 


.9550 


73.0 


34.9 


38.1 


.9820 


35.2 


21.8 


13.4 


.9540 


73.8 


35.3 


38.5 


.9810 


36.7 


22.3 


14.4 


.9530 


74.6 


35.6 


39.0 


.9800 


38.3 


22.8 


15.5 


.9520 


75.4 


35.9 


39.5 


.9790 


39.9 


23.4 


16.5 


.9510 


76.2 


36.2 


40.0 


.9780 


41.5 


24.0 


17.5 


.9500 


76.9 


36.5 


40.4 


.9770 


43.1 


24.5 


18.6 


.9490 


77.6 


36.8 


40.8 


.9760 


44.8 


25.0 


19.8 


.9480 


78.3 


37.0 


41.3 


.9750 


46.5 


25.5 


21.0 


.9470 


79.0 


37.3 


41.7 


.9740 


48.2 


26.0 


22.2 


.9460 


79.7 


37.6 


42.1 


.9730 


49.8 


26.5 


23.3 











Scale readings are applicable only to instruments calibrated in arbitrary scale units proposed by Pulfrich, Z angew. Chem., 1899, p. 1168. According 
to this scale, 14.5 - 1.33300, 50.0 = 1.34650, and 100.0 = 1.36464. If instrument used is calibrated in other arbitrary units, refractive index corresponding 
to observed reading can be converted into equivalent Zeiss reading by referring to 52.004 



158 



9. Beverages; Distilled Liquors 



AOAC Methods (1980) 



9.093 



Determination 



Inject 10 fxV. MeOH std soln. Adjust operating parameters and 
attenuation to obtain measurable peak ht (ca % full scale 
deflection). Det. retention time of MeOH and /7-BuOH (ca 3 and 
12 min, resp.). Inject 10 (jlL sample to est. MeOH, using 'atten- 
uation if necessary, and to check for absence of /?-BuOH. On 
basis of presence or absence of a?-BuOH in sample, det. MeOH 
content from std curve prepd according to (a) or (b): 

(a) n-Butyl alcohol absent. — On basis of est. of MeOH, prep, 
series of stds (4 or 5) in which range of concn includes MeOH 
concn in sample. Add internal std to both sample and std solns 
at concn similar to that of MeOH in sample. Calc. peak ht ratios 
of MeOH:n-butyl alcohol, using av. of duplicate injections, and 
plot ratios against MeOH concn. 

(b) n-Butyl alcohol present. — Prep, series of MeOH stds as in 
(a), but do not add n-BuOH to sample or stds. Plot actual peak 
ht of MeOH against concn. 

Artificial Colors 
(See also 9.005-9.008) 

9.094 Marsh Test — Official First Action 

To 10 ml_ sample in 20 ml_ test tube add enough freshly 
shaken Marsh reagent (100 mL amyl alcohol, 3 mL H 3 P0 4 , and 
3 mL H 2 0) to nearly fill tube, and shake several times. Let layers 
sep. Color in lower layer indicates that sample has been colored 
with caramel, synthetic dye, or extractive material from un- 
charred white oak chips. 

In absence of any color, test 10 mL in same manner, using 
enough fusel oil, amyl alcohol, or Pentasol to nearly fill tube 
and shaking several times. Deeply colored lower layer indicates 
synthetic dye. Det. its identity as in Chapter 34. To confirm 
caramel apply one or more of following tests: 

9.095 Mathers Test — Official Final Action 
See 11.055. 

9.096 * Cyclohexanol Test — Official Final Action it 
See 9.059, 10th ed. 

9.097 Coal-Tar Colors 
See Chapter 34. 



mL Na 2 C0 3 soln, and dil. to vol. with H 2 0. Mix well and det. A 
after 30 min at 760 nm. Plot>4 against mg tannic acid/100 mL. 



Tannin (22) — Official Final Action 



9.098 



Reagents 



(a) Folin-Denis reagent.— -To 750 mL H 2 add 100 g Na 2 W0 4 
.2H 2 0, 20 g phosphomolybdic acid, and 50 mL H 3 P0 4 . Reflux 2 
hr, cool, and dil. to 1 L. 

(b) Sodium carbonate saturated soln. — To each 100 mL H 2 
add 35 g anhyd. Na 2 C0 3 , dissolve at 70-80°, and let cool 
overnight. Seed supersatd soln with crystal of Na 2 CO 3 .10H 2 O, 
and after crystn filter thru glass wool. 

(c) Tannic acid std soln. — 0.1 mg/mL. Dissolve 100 mg tannic 
acid in 1 L H 2 0. Prep, fresh soln for each detn. 



9.099 



Preparation of Standard Curve 



Pipet 0-10 mL aliquots std tannic acid soln into 100 mL vol. 
flasks contg 75 mL H 2 0. Add 5 mL Folin-Denis reagent and 10 

it Surplus method — see inside front cover. 



9.100 



Determination 



Using 1 mL sample, det. A as in 9.099 and obtain mg tannic 
acid/100 mL from std curve. If A is too great, repeat detn on 
1 +4 diln of sample. Samples treated as above may be compared 
in Nessler tubes against freshly prepd tannic acid stds treated 
in same manner. 



Cyanide (23)— Official First Action 



9.101 



Apparatus 

(Letters refer to Fig. 9:04.) 

(a) Distilling flask. — 500 mL, r-b, 3-neck with ~$ joints, angle- 
type (Fisher Scientific Co., No. 10-165B, or equiv.) (A). 

(b) Thermometer. — Range -10 to 110°, with f screw-cap 
adapter to fit one f side neck of distg flask (B). 

(c) Air inlet tube. — With f cone to fit side neck of distg flask 
(C). 

(d) Condenser. — Graham coil-type, with inner and outer $ 
joints, f cone to fit center neck of distg flask (Fisher Scientific 
Co., No. 7-728B, 400 mm jacket length, or equiv.) (D). 

(e) Flange assembly. — Consisting of 2 sep. ground-glass 
flanges at one end of sep. glass tubing, 7 mm diam. Lower tube 
(E) is ca 14 mm long and is also fitted with ? cone to fit $ socket 
at top of condenser; f cone has 2 glass hooks. Upper tube (F) 
is ca 5 mm long. 

(f) Clip.— To connect 2 ground-glass flanges together (G). 

(g) Springs. — To connect (D) and (F) securely together (H). 




FIG. 9:04 — Apparatus for determining hydrogen cyanide in distilled 

spirits. 



AOAC Methods (1980) 



Cordials and Liqueurs 



159 



9.102 Reagents 

(a) Potassium ferrocyanide so/n. — (7) Stock soln. — 0.01% 
K 4 Fe{CN) 6 .3H 2 in 0.2% Na 2 C0 3 . Store in amber bottle. Na 2 C0 3 
stabilizes this soln. (2) Working so/n .—0.0001% K 4 Fe(CN) 6 .3H 2 0; 
27 mL equiv. to 10 /xg CN. Prep, fresh daily by dilg 1.0 mL stock 
soln to 100 mL with H 2 0. 

( b ) 4,4 '-Meth ylenebis (A/, N- dimeth yla nil in e ) {tetrabase ) . — 
Eastman Kodak Co., No. 244; or equiv. 

(c) Bis{ethyl acetoacetato) copper {copper ethyl acetoace- 
tate). — Fisher Scientific Co., No. 10057; or equiv. 

(d) Color reagent. — Dissolve 50 mg tetrabase and 50 mg Cu 
Et acetoacetate in 10 mL CHCI 3 . Store in closed bottle; prep, 
fresh weekly. 

(e) Test papers. — Whatman No. 44 paper, or equiv., cut to 
size in strips to fit between flanges of ground-glass flange 
assembly. Impregnate with several drops color reagent imme- 
diately before use; let CHCI 3 evap. completely before inserting 
test paper between flanges of assembly. 

(f) Lead dioxide.— Dissolve ca 40 g Pb{OAc) 2 in 200 mL H 2 
by heating on H 2 bath in hood. Add NaOCI (13% available CI) 
until there appears to be no further darkening in color (ca 100 
mL). Ppt should be dark brown at this stage. Let stand ca 15 
min. Filter thru double thickness Whatman No. 42 papers in 
buchner. Wash with H 2 0, discard filtrate, and wash ppt with ca 
200 mL HN0 3 (1+9). Finally wash with H 2 and dry overnight 
in 105° oven. Pb0 2 produced by this method is granular and 
should be broken into pieces ca 20 mesh, but must not be 
ground any finer. Check each batch by applying control test for 
ferrocyanide std, 11.057(a), on 100 mL aliquots of H 2 contg 10 
HQ CN and 500 ppm S0 2 (as NaHS0 3 ) and comparing stain 
produced with std stain. Anal, grade Pb0 2 is not suitable because 
it is too fine and prevents free flow of N to test papers. 

9.103 Distilled Spirits Free of Sulfur Dioxide 

(For products contg S0 2 , see 11.057.) 

(a) Control test.— Add 27 mL K 4 Fe(CN) 6 .3H 2 working soln 
(equiv. to 10 ^9 CN) to 100 mL alcohol and 100 mL H 2 in 500 
mL 3-neck flask with stopper in 1 side neck and gas inlet tube 
connected to N (or C0 2 ) in other. (Use of gas to flush HCN thru 
test papers is preferred, but vac. may be used.) Connect center 
neck of flask to condenser and place ground-glass flange as- 
sembly in top of condenser; then connect hooks with springs. 
Insert test paper between 2 glass flanges and clip together. 
Remove stopper momentarily, add 10 mL H 2 S0 4 (1+9), and 
immediately replace stopper. 

Assure rapid flow of cold H 2 thru condenser and then heat 
contents of flask just to bp. When boiling temp, is reached, pass 
N thru liq. at rate such that individual bubbles just cease to be 
visible as sep. bubbles, but form continuous stream. Check that 
all joints are tight. Let distn proceed 15 min; then remove test 
paper and examine for blue stain which indicates cyanide. 
(Alcohol vapors mustnot reach test paper.) Intense, well defined 
blue circle should be obtained with 10 fxg CN. Blank detn 
performed concurrently must show no color. (1 /xg CN, equiv. 
to 0.01 ppm CN in 100 mL sample, can be readily detected.) 

(b) Samples— Add 100 mL sample to 100 mL H 2 in 500 mL 
3-neck distg flask, and test as in (a). For pos. results, confirm 
test papers showing faint or questionable stains by 2 addnl 
analyses. Blue stains are stable ca 1 week if kept out of direct 
light. 

(c) Confirmation. — To confirm that pos. reactions from sam- 
ples of unknown origin are due to CN, expose blue test paper 
to NH 3 vapors. Colorless carbinol base is formed. With addn of 
HOAc, blue reappears. 



CORDIALS AND LIQUEURS 



9.104 Physical Examination — Procedure 

Note and record following: {a) Appearance, whether bright or 
turbid and presence of sediment; ib) color and depth of color; 
(c) odor; {d) taste. 

9.105 Specific Gravity — Official Final Action 
See 9.011. 

9.106 Alcohol — Official Final Action 

(a) By weight— See 9.021. 

(b) By volume. — See 9.013. Use pycnometer calibrated at 
15.56°. 



Methanol — Official Final Action 

9. 1 07 Preparation of Sample 

Measure sample contg 20-25 mL absolute alcohol into distg 
flask, add enough H 2 to make total ca 100 mL, and distil, 
collecting ca 50 mL distillate. To distillate add 4 g NaCI for each 
10 mL H 2 and let stand several hr for complete satn. 

Transfer to separator, using ca 10 mL satd NaCI soln to wash 
out container, and shake with 25 mL pet ether. When sepn is 
complete, transfer aq. soln to second separator contg 25 mL pet 
ether; shake, and transfer aq. soln to third separator, also contg 
25 mL pet ether; shake, and when sepn is complete, drain aq. 
soln into 200 mL distg flask. Meanwhile add 25 mL satd NaCI 
soln to first separator and follow sample thru with this soln, 
finally adding washings to sample soln in distg flask. Repeat 
this operation with second 25 mL portion satd NaCI soln, finally 
adding this also to distg flask. Distil mixt. into 50 mL vol. flask, 
using suitable adapter. After 48-49 mL distils, disconnect app., 
fill flask to mark with H 2 0, mix, and det. MeOH as in 9.089 or 
9.090. 

9.108 Aldehydes—Official Final Action 

Measure 100-200 mL sample into distn flask. If solid content 
is =£25 g/100 mL, add 12.5-25 mL H 2 0; if >25 g/100 mL, add 5 
mL H 2 for each 10 g solids present; distil slowly, collecting vol. 
distillate equal to that of sample, and proceed as in 9.052. 

9.109 Fusel Oil — Official Final Action 

Using 50 mL prepd distillate, 9.108, proceed as in 9.068, 
9.071-9.074, or 9.077. 

9.110 Total Solids—Official Final Action 

(a) From specific gravity of dealcoholized sample. — Transfer 
residue from alcohol detn, 9.106(b), to original pycnometer with 
H 2 0, dil. to mark with H 2 at 15.56°, and mix. Adjust temp, of 
pycnometer and contents to 20°; adjust meniscus to mark, using 
capillary tube or narrow strips of filter paper to remove any 
excess liq. while in 20° bath. Weigh, and calc. sp gr of liq. From 
52.008 det. % dry substance and corresponding sp gr at 20°/4°. 
Sp gr at 20°/4° x % dry substance = total solids (g/100 mL). 

(b) By evaporation. — Fill 25 mL vol. flask with sample at 20°, 
and adjust meniscus, using capillary tube or narrow strips of 
filter paper, while flask is immersed in bath held at same temp, 
ca 30 min. Quant, transfer contents of flask to 100 mL vol. flask 
with H 2 and dil. to vol. with H 2 at convenient temp. At same 
temp., pipet 10 mL dild sample into dish contg sand and dry as 
in 31.008. Wt residue x 40 = total solids (g/100 mL). 



160 



9. Beverages: Distilled Liquors 



AOAC Methods (1980) 



(c) From refractive index of deaicohoiized sample. — Restore 
residue from alcohol detn to original vol. by evapg or dilg as 
necessary. Det. refractometer reading of soln at 20° and obtain 
corresponding % dry substance. From 52.008 det. sp gr corre- 
sponding to % dry substance found and multiply by % dry 
substance to obtain g total sol ids/ 100 mL sample. To obtain 
% total solids, divide total sol ids/ 100 mL by sp gr, 9.011. 

9.111 * Glycerol — Official Final Action * 

By direct weighing, or by oxidn with K 2 Cr 2 7 soln. See 
9.111,12th ed. 

9.112 Sucrose — Official Final Action 

(a) By polarization. — Pipet, into evapg dish, vol. sample equiv. 
to 52 g as calcd from sp gr, 9.011, and exactly neutze with 1/V 
NaOH, calcg amt required from acidity, 9.119. Evap. on steam 
bath to remove alcohol, transfer to 200 mL vol. flask, and 
proceed as in 31.025 or 31.026, beginning ". . . add necessary 
clarifying agent, . . ." in 31.025(a). 

(b) By reducing sugars before and after inversion. — Approx- 
imate sugar content of sample from total solids, 9.1 10, and pipet 
sample contg 5-7 g sugars into porcelain dish; exactly neutze 
with 1/V NaOH soln, calcg amt required from acidity, 9.119, and 
evap. on steam bath to remove alcohol. Transfer to 200 mL vol. 
flask, clarify with neut. Pb(OAc) 2 soln, 31.021(d), remove excess 
Pb with K oxalate, and proceed as in 31.031, using 31.038 for 
detn of reducing sugars. 

9.113 Ash— Official Final Action 

Proceed as in 31.012 or 31.013, using 25 mL sample. 

9.114 Soluble and Insoluble Ash — Official Final Action 

Using ash from 9.113, proceed as in 31.015. 

9.115 Alkalinity of Soluble Ash— Official Final Action 

Using sol. ash from 9.114, proceed as in 31.016. 

9.116 Alkalinity of Insoluble Ash — Official Final Action 

Using insol. ash from 9.114, proceed as in 31.017. 

9.117 Phosphorus — Official Final Action 

Using ash obtained in 9.113, det. P 2 5 as in 11.034. 

9.118 Caramel — Official Final Action 
See 11.055. 

9.119 Total Acidity — Official First Action 

Place ca 600 mL H 2 in 800 mL beaker, add ca 1 mL phthln, 
and titr. to pink soln with 0.1/V NaOH. Add 10-20 mL sample 
(unless this vol. gives soln such deep color that it will obscure 
end point, in which case 5 mL may be used) and titr. to pink 
comparable to that of soln before sample was added. Calc. 
acidity as g/100 mL sample in terms of predominating acid 
present in sample. 

9.120 Characteristic Acids — Preparation 
of Sample — Procedure 

Use sample contg =s30 g solids and =e200 mg acid to be detd, 
as calcd from acidity; evap. to ca 30 mL and treat as in 
9.121-9.124. 

* Surplus method— see inside front cover. 



9.121 if Tartaric Acid — Official Final Action if 
See 9.121, 12th ed. 

9.122 * Citric Acid— Official Final Action * 
See 9.122, 12th ed. 

9.123 if Total Malic Acid (Laevo and Inactive) * 

Official First Action 

See 9.123, 12th ed. 

9.124 * Laevo-Malic Acid — Official First Action if 

See 9.124, 12th ed. 

9.125 Volatile Esters — Official Final Action 

Measure 100-500 mL sample into distg flask and steam distil 
as in 12.026, collecting vol. distillate at least twice as great as 
vol. alcohol contained in sample. (If detn 9.126 is to be made, 
use 500 mL sample.) Disconnect app. and wash out condenser 
with little H 2 0. Add ca 1 mL phthln, and titr. to pink that persists 
>1 min, using 0.1/V NaOH or KOH. Add measured excess of 
25-50 mL 0.1/V alkali to soln, reflux 1 hr, cool, and titr. excess 
alkali with 0.1/V H 2 S0 4 . Calc. number of mL 0.1/V alkali used in 
saponification of esters as EtOAc. 1 mL 0.1/V alkali = 8.8 mg 
EtOAc. 

9.126 * Gamma Undecalactone if 

(Qualitative Test) (24) 
Official Final Action 

(Peach and apricot cordials) 

See 9.087, 10th ed. 

9.127 if Optical-Crystallographic Properties if 

of Hydrazino-y-Undecalactone 
Official Final Action 

See 9.088, 10th ed. 

9.128 Benzaldehyde — Official Final Action 
See 19.100-19.101. 

9.129 Thujone (25)— Official First Action 

To 500 mL sample add 1 mL freshiy distd aniline and 1 mL 
H3PO4, and reflux 30 min on steam bath. Distil two 100 mL 
portions; reject first and test second for thujone as follows: 

Add 0.5 g semicarbazide hydrochloride and 0.6 g anhyd. 
NaOAc (or 1.0 g crystd salt) and let mixt. stand overnight. Distil 
off alcohol at min. pressure. Steam distil to remove essential 
oils and other volatile material; collect and reject first ca 15 mL 
distillate. Wash down condenser with little alcohol and with 
H 2 0. Cool sample, add 1 mLH 2 S0 4 (1 + 1), and again steam distil, 
collecting 20 mL distillate in cylinder. Pour distillate into small 
separator, and add 20 mL ether, using receiver as measure. 
Shake and sep. ether soln. Add 10 mL 65% alcohol and let ether 
evap. spontaneously. After all ether evaps, note odor of residue. 
Odor of thujone will be apparent if 5=2 mg is present in soln, 
provided it is not masked by presence of other odoriferous 
substances. Make modified Legal test as follows: 

To soln obtained as above, add 1 mL 10% ZnSO A soln and 
0.25 mL freshly prepd aq. Na nitroprusslde soln (0.1 g/mL). 
Slowly, with const stirring, add 2 mL 5% NaOH soln. Let stand 
1-2 min. Add 1.5 mL HOAc and mix. Ppt of raspberry red color 
(resembling alcohol ppt of red fruit juice) shows presence of 
thujone. Neg. test is shown by similar ppt having appearance 



AOAC Methods (1980) 



Selected References 



161 



similar to that of alcohol ppt from apple jelly or other light 
colored fruit. 

SELECTED REFERENCES 

(/) JAOAC 39, 723(1956); 41, 118(1958). 

(2) JAOAC 38, 821(1955); 39, 730(1956); 40, 440(1957). 

(3) Ind. Eng. Chem., Anal. Ed. 14, 237(1942); JAOAC 28, 
88(1945); 41, 118(1958); 42, 329(1959). 

(4) JAOAC 40, 436(1957); 42, 327(1959); 43, 657(1960). 

(5) Ind. Eng. Chem. 18, 841(1926); JAOAC 35, 239(1952). 

(6) JAOAC 37, 945(1954); 46, 299(1963); 47, 720(1964). 

(7) JAOAC 50, 338(1967). 
{8) JAOAC 50, 334(1967). 
(9) JAOAC 53, 12(1970). 

(10) JAOAC 49, 498(1966). 
(7 7) JAOAC 37, 921(1954). 



(72) JAOAC 55, 559(1972). 

(73) JAOAC 55, 566(1972). 

(14) JAOAC 42, 331(1959); 43, 655(1960); 44, 383(1961). 

(15) JAOAC 46, 285(1963). 

(16) JAOAC 51, 915(1968). 
(77) JAOAC 55, 549(1972). 

(75) JAOAC 43, 659(1960); 44, 392(1961). 

(19) JAOAC 41, 121(1958); 42, 336(1959). 

(20) J. Am. Chem. Soc. 27, 964(1905); Ind. Eng. Chem. 19, 
844(1927); JAOAC 28, 800(1945). 

(21) JAOAC 55, 564(1972). 

(22) JAOAC 35, 255(1952); 37, 665(1954). 

(23) JAOAC 53, 777(1970). 

(24) JAOAC 16, 420(1933); 19, 75, 183(1936). 

(25) Ann. chim. anal. 13, 227(1908); Schweiz. Wochschr. 49, 
337, 507(1911); JAOAC 19, 120(1936); 20, 69(1937). 



10. Beverages: Malt Beverages and Brewing Materials* 



BEER 

(Unless otherwise directed, express results as % by wt.) 

10.001 Preparation of Sample 
Official Final Action 

Remove C0 2 by transferring sample to large flask and shaking, 
gently at first and then vigorously, keeping temp, of beer at 
20-25°. If necessary, remove suspended material by passing the 
C0 2 -free beer thru dry filter paper. 

Color 

Spectrophotometry Method (Standard 

Reference Color Method) (?) 

Official Final Action 

10.002 Apparatus 

Spectrophotometer. — Capable of isolating band width of =s1 
nm at 430 nm with wavelength and photometer scales checked 
and corrected for inaccuracies in accordance with instructions 
contained in NBS Letter Circular LC-1017 of Jan. 1967. 

10.003 Preparation of Sample 

Partially degas sample by opening bottle at room temp., 
pouring contents into 1 L erlenmeyer, and swirling gently. Avoid 
formation of turbidity, and conduct partial degassing and read- 
ings as rapidly as possible. 



10.004 



Determination 



Place prepd sample in suitable cell and det. A at 430 nm and 
at 700 nm. 



10.005 



Calculations 



Calc. A from thickness at which read to 1 / 2 " (1.27 cm) (4y 2 ). If 
(>4tt at 43onm) x 0.039 > (4 Vi at 700 nm), sample is assumed "free of 
turbidity" and color is calcd as follows: 

Beer color intensity = 10 x (/4y 2 at430nm ). If [Ay laX7Q0nm ) > 0.039 
x (/4Ya a t43onm), clarify sample by centrfg or filtering, and redet. 
A. 

Report color intensity values to nearest 0.1 unit. 



Photometric Method — Official Final Action 



10.006 



Apparatus 

Use any com. available filter photometer or abridged spec- 
trophtr utilizing moderately broad spectral band and having 
adequate sensitivity. Use light filter with peak T in range 420-450 
nm (blue-violet) for max. sensitivity and precision. (Filters for 
wavelengths in blue or blue-green range may also be used, but 
result in reduced precision.) Cell should be of such size, if 
possible, as to give A values between 0.187 and 0.699 (20-65% 
T), where max. precision is achieved. Use same size cell for 
both color measurement and calibration. 

10.007 Calibration of Photometers 

Beer calibration method. — For each color intensity value for 



which measurements are to be made, obtain 6-8 replicate 
bottles of beer which are low in air content and have been 
pasteurized. 

Det. color intensity value of the beer by averaging readings 
obtained for ^2 bottles by Standard Reference Color {SRC) 
Method, 10.004. If these values must be obtained from another 
laboratory, ship bottles of beer by the fastest available method, 
marked to avoid rough handling. 

Det. photometer reading of the beer by averaging readings 
obtained for 5=2 bottles with wavelength and cell as in 10.006. 
Calc. calibration factor in accordance with photometer instruc- 
tions or prep, calibration curve by plotting A or photometer 
scale reading against the SRC value for sample, assuming that 
curve passes thru origin. This calibration will be accurate only 
for readings in immediate vicinity of calibration point. If it is 
desired to accurately measure color intensity of >1 sample or 
colors over range of values, calibrate photometer for each 
sample or use beers having colors which cover desired range. 
Calc. av. calibration factor or prep. av. calibration curve. 



10.008 
See 10.003. 

10.009 



Preparation of Sample 



Determination 



Place sample in cell and det. photometer reading. Calc. color 
intensity value, using calibration factor or calibration curve. 
Report color to nearest 0.1 unit. 

Total Haze after Chilling (2)— Official Final Action 
10.010 Reagents 

(Use turbidity-free distd H 2 thruout.) 

(a) Hydrazine sulfate soln—Wo. Dissolve 1.000 g H 4 N 2 .H 2 S0 4 
in H 2 (may require 4-6 hr) and dil. to 100 mL. 

(b) Stock formazin suspension. — Dissolve 2.500 g hexame- 
thylenetetramine (formin) in 25 mL H 2 in 125 mL erlenmeyer, 
pipet in 25 mL 1% hydrazine sulfate soln, and stopper flask. 
Formazin begins to ppt in 6-8 hr and pptn is complete within 
24 hr. Prep, every 3 months. 

(c) 1000 Turbidity std.—DW. 14.5 mL well mixed stock sus- 
pension, (b), to 1 L with H 2 in vol. flask. Prep, weekly. (1000 
formazin turbidity units (FTU) on empirical formazin turbidity 
scale represents reflectance of insol. reaction products of 0.0725 
g hydrazine sulfate with 0.7250 g hexamethylenetetramine dild 
to 1 L) 

(d) Working stds for visual method.— Prep, suitable dilns, 
daily, of 1000 turbidity std with H 2 0. FTU stds <100 are suitable 
for fresh beers; higher stds may be required for older samples. 
Use increments of 10 FTU for stds <100 FTU; in 20 FTU 
increments for 100-200; and in 50 FTU increments for >200 
FTU. 



Visual Method 



10.011 



Apparatus 

(a ) Clark Turbidimeter, Model CL T. — Code 648. Ava i I a b I e f ro m 
Cargille Scientific Inc., 55 Commerce Rd, Cedar Grove, NJ 07009. 



* Many methods in this chapter have been tested by both American Society of Brewing Chemists and Association of Official Analytical Chemists and 
have been adopted by both Associations. See "Methods of Analysis, A.S.B.C.," 7th rev. ed., 1976. 



162 



AOAC Methods (1980) 



Beer 



163 



Viewing box of same dimensions and lighting is also suitable. 

(b) Red Plexiglas sheet— V* thick, ca 1 sq ft (930 sq cm). 

(c) Constant temperature bath. — 0±0.2°. 

(d) Ice-water bath. — Contg few drops wetting agent. 

(e) Flint glass bottles. — Of same dimensions as flint glass 
bottles contg beer test samples; or clear drinking glasses (shells), 
10 oz (300 mL), od ca 66 mm at bottom and 67 mm at top. 

10.012 Determination 

(Make comparisons with samples at 0°. Keep test samples in 0° 
bath when not matching turbidities.) 

Place container of beer to be tested in upright position in 0° 
bath and hold 24 hr. 

Prep, series of formazin turbidity working stds covering range 
of expected turbidities of test samples. Fill into flint glass bottles 
of same dimensions as those holding beer test samples. 

If beer is in flint glass bottles, carefully remove bottle from 
const temp, bath without disturbing sediment. Dip bottle into 
ice-H 2 bath contg few drops wetting agent to prevent fogging 
or accumulation of H 2 droplets on bottle while in viewing box. 
Place bottle of beer in viewing box between 2 bottles of formazin 
turbidity working stds. Compare turbidities by viewing thru red 
Plexiglas sheet placed 5 cm (2") in front of bottles. Change 
formazin stds until that working std is found which most closely 
matches turbidity of test sample. 

If beer is not in flint glass bottles, carefully remove container 
from const temp, bath and, without disturbing sediment, pour 
beer into clear 10 oz drinking glass (shell) which has been 
prechilled by standing (external contact only) in ice-H 2 bath 
contg wetting agent. Degassing is not necessary. Use formazin 
turbidity working stds in identical 10 oz glasses to match 
turbidities as above for bottles. 

Report as total haze of the beer after chill ing, formazin turbidity 
units (FTU) of working std giving closest match. In range 0-100 
FTU, report to nearest 10 FTU; 100-200, 20; >200, 50. 



Nephelometric Method 



10.013 



Calibration 



Nephelometer. — Prep, calibration curve at 580 nm or other 
suitable wavelength for instrument employed by use of series 
of working stds or dilns of 1000 turbidity std. If readout device 
of nephelometer is 0-100 scale of arbitrary units, set needle to 
indicate units when cell is filled with turbidity-free distd H 2 
and 100 units when it is filled with selected formazin turbidity 
std. 



10.014 



Determination 



Place containers of beer to be tested in upright position in 0° 
bath and hold 24 hr. 

Prechill nephelometer cell in small ice~H 2 bath contg wetting 
agent (external contact only). Carefully remove container of beer 
from bath and, without disturbing sediment, rinse and fill cell 
with test sample. Place cell in ice-H 2 bath contg wetting agent, 
and degas beer by stirring with thermometer. When beer temp, 
is 0°, place cell in sample chamber of nephelometer and det. 
reading. (Beer must be at 0° when taking reading.) 



10.015 



Calculations 



Calc. FTU total haze from calibration curve or by formula: 
FTU = R x 5/100, where R = nephelometer (galvanometer 
scale) reading; S = FTU of formazin turbidity std used for 
calibration of nephelometer. 



10.016 Specific Gravity — Official Final Action 

Det. sp gr of prepd sample, 10.001, at 20/20° (in air) as in 
9.011, but use pycnometers described in 10.106(i) and 10.107(b) 

or (c). 



Viscosity of Beer [3) — Official Final Action 



10.017 



Apparatus 

(a) Constant temperature bath. — Adjusted to 20.00 ±0.05°. 

(b) Viscometer. — Ostwald or Cannon-Fenske; H 2 time range 
50-150 sec. 



10.018 



Determination 



Prep, sample as in 10.001, ensuring complete decarbonation 
but avoiding loss of surface active components. Det. sp. gr. as 
in 10.016. 

Clean viscometer with chromic acid cleaning soln, rinse with 
H 2 0, and drain. Attemperate viscometer, H 2 0, and beer to 20° 
in H 2 bath. Add appropriate vol. H 2 (usually 5 or 10 mL) at 20° 
to viscometer. Using suction, draw H 2 above upper mark. Let 
liq. level fall, and start timing with stopwatch as meniscus 
passes upper mark. Stop timing when meniscus passes lower 
mark. 

Rinse viscometer with beer sample, and det. time required for 
passage of beer between menisci as above. 

1 0.019 Calculations 

(a) Absolute or dynamic viscosity {Centi poise). — (7) 
Viscosity (cP) = flow time of beer at 20° x sp gr of beer 

x 1.002/flow time of H 2 at 20°, 
where 1.002 = viscosity of H 2 in